Note: Descriptions are shown in the official language in which they were submitted.
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ISOLATED HUMAN TRANSPORTER PROTEINS, NUCLEIC ACID
MOLECULES ENCODING HUMAN TRANSPORTER PROTEINS,
AND USES THEREOF
RELATED APPLICATIONS
The present application claims priority to U.S. Serial No. 09/752,821, filed
January 3, 2001 (Atty. Docket CL001065) and U.S. Serial No. 09/861,846, filed
May
22, 2001 (Atty. Docket CL001065CIP).
FIELD OF THE INVENTION
The present invention is in the field of transporter proteins that are related
to
the sodium/chloride-dependent organic solute cotransporter subfamily,
recombinant
DNA molecules, and protein production. The present invention specifically
provides
novel peptides and proteins that effect ligand transport and nucleic acid
molecules
encoding such peptide and protein molecules, all of which are useful in the
development of human therapeutics and diagnostic compositions and methods.
BACKGROUND OF THE INVENTION
Transporters
Transporter proteins regulate many different functions of a cell, including
cell
proliferation, differentiation, and signaling processes, by regulating the
flow of
molecules such as ions and macromolecules, into and out of cells. Transporters
are
found in the plasma membranes of virtually every cell in eukaryotic organisms.
Transporters mediate a variety of cellular functions including regulation of
membrane
potentials and absorption and secretion of molecules and ion across cell
membranes.
When present in intracellular membranes of the Golgi apparatus and endocytic
vesicles, transporters, such as chloride channels, also regulate organelle pH.
For a
review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.
Transporters are generally classified by structure and the type of mode of
action. In addition, transporters are sometimes classified by the molecule
type that is
transported, for example, sugar transporters, chlorine channels, potassium
channels,
etc. There may be many classes of channels for transporting a single type of
molecule
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(a detailed review of channel types can be found at Alexander, S.P.H. and J.A.
Peters:
Receptor and transporter nomenclature supplement. Trends Pharmacol. Sci.,
Elsevier,
pp. 65-68 (1997)).
The following general classification scheme is known in the art and is
followed in the present discoveries.
Channel-type transporters. Transmembrane channel proteins of this class are
ubiquitously found in the membranes of all types of organisms from bacteria to
higher
eukaryotes. Transport systems of this type catalyze facilitated diffusion (by
an energy-
independent process) by passage through a transmembrane aqueous pore or
channel
without evidence for a carrier-mediated mechanism. These channel proteins
usually
consist largely of a-helical spanners, although b-strands may also be present
and may
even comprise the channel. However, outer membrane porin-type channel proteins
are
excluded from this class and are instead included in class 9.
Carrier-type transporters. Transport systems are included in this class if
they
utilize a carrier-mediated process to catalyze uniport (a single species is
transported
by facilitated diffusion), antiport (two or more species are transported in
opposite
directions in a tightly coupled process, not coupled to a direct form of
energy other
than chemiosmotic energy) and/or symport (two or more species are transported
together in the same direction in a tightly coupled process, not coupled to a
direct
form of energy other than chemiosmotic energy).
Pyrophosphate bond hydrolysis-driven active transporters. Transport systems
are included in this class if they hydrolyze pyrophosphate or the terminal
pyrophosphate bond in ATP or another nucleoside triphosphate to drive the
active
uptake and/or extrusion of a solute or solutes. The transport protein may or
may not
be transiently phosphorylated, but the substrate is not phosphorylated.
PEP-dependent, phosphoryl transfer-driven group translocators. Transport
systems of the bacterial phosphoenolpyruvateaugar phosphotransferase system
are
included in this class. The product of the reaction, derived from
extracellular sugar, is
a cytoplasmic sugar-phosphate.
Decarboxylation-driven active transporters. Transport systems that drive
solute (e.g., ion) uptake or extrusion by decarboxylation of a cytoplasmic
substrate are
included in this class.
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Oxidoreduction-driven active transporters. Transport systems that drive
transport of a solute (e.g., an ion) energized by the flow of electrons from a
reduced
substrate to an oxidized substrate are included in this class.
Light-driven active transporters. Transport systems that utilize light energy
to
drive transport of a solute (e.g., an ion) are included in this class.
Mechanically-driven active transporters. Transport systems are included in
this class if they drive movement of a cell or organelle by allowing the flow
of ions
(or other solutes) through the membrane down their electrochemical gradients.
Outer-membrane porins (of b-structure). These proteins form transmembrane
pores or channels that usually allow the energy independent passage of solutes
across
a membrane. The transmembrane portions of these proteins consist exclusively
of b-
strands that form a b-barrel. These porin-type proteins are found in the outer
membranes of Gram-negative bacteria, mitochondria and eukaryotic plastids.
Methyltransferase-driven active transporters. A single characterized protein
currently falls into this category, the Na+-transporting
methyltetrahydromethanopterin:coenzyme M methyltransferase.
Non-ribosome-synthesized channel-forming peptides or peptide-like
molecules. These molecules, usually chains of L- and D-amino acids as well as
other
small molecular building blocks such as lactate, form oligomeric transmembrane
ion
channels. Voltage may induce channel formation by promoting assembly of the
transmembrane channel. These peptides are often made by bacteria and fungi as
agents of biological warfare.
Non-Proteinaceous Transport Complexes. Ion conducting substances in
biological membranes that do not consist of or are not derived from proteins
or
peptides fall into this category.
Functionally characterized transporters for which sequence data are lacking.
Transporters of particular physiological significance will be included in this
category
even though a family assignment cannot be made.
Putative transporters in which no family member is an established transporter.
Putative transport protein families are grouped under this number and will
either be
classified elsewhere when the transport function of a member becomes
established, or
will be eliminated from the TC classification system if the proposed transport
function
is disproven. These families include a member or members for which a transport
function has been suggested, but evidence for such a function is not yet
compelling.
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Auxiliary transport proteins. Proteins that in some way facilitate transport
across one or more biological membranes but do not themselves participate
directly in
transport are included in this class. These proteins always function in
conjunction
with one or more transport proteins. They may provide a function connected
with
energy coupling to transport, play a structural role in complex formation or
serve a
regulatory function.
Transporters of unknown classification. Transport protein families of
unknown classification are grouped under this number and will be classified
elsewhere when the transport process and energy coupling mechanism are
characterized. These families include at least one member for which a
transport
function has been established, but either the mode of transport or the energy
coupling
mechanism is not known.
Ion channels
An important type of transporter is the ion channel. Ion channels regulate
many different cell proliferation, differentiation, and signaling processes by
regulating
the flow of ions into and out of cells. Ion channels are found in the plasma
membranes of virtually every cell in eukaryotic organisms. Ion channels
mediate a
variety of cellular functions including regulation of membrane potentials and
absorption and secretion of ion across epithelial membranes. When present in
intracellular membranes of the Golgi apparatus and endocytic vesicles, ion
channels,
such as chloride channels, also regulate organelle pH. For a review, see
Greger, R.
(1988) Annu. Rev. Physiol. 50:111-122.
Ion channels are generally classified by structure and the type of mode of
action. For example, extracellular ligand gated channels (ELGs) are comprised
of
five polypeptide subunits, with each subunit having 4 membrane spanning
domains,
and are activated by the binding of an extracellular ligand to the channel. In
addition,
channels are sometimes classified by the ion type that is transported, for
example,
chlorine channels, potassium channels, etc. There may be many classes of
channels
for transporting a single type of ion (a detailed review of channel types can
be found
at Alexander, S.P.H. and J.A. Peters (1997). Receptor and ion channel
nomenclature
supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 and http://www-
biology.ucsd.edu/~msaier/transport/toc.html.
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There are many types of ion channels based on structure. For example, many
ion channels fall within one of the following groups: extracellular ligand-
gated
channels (ELG), intracellular ligand-gated channels (ILG), inward rectifying
channels
(INR), intercellular (gap junction) channels, and voltage gated channels
(VIC). There
5 are additionally recognized other channel families based on ion-type
transported,
cellular location and drug sensitivity. Detailed information on each of these,
their
activity, ligand type, ion type, disease association, drugability, and other
information
pertinent to the present invention, is well known in the art.
Extracellular ligand-gated channels, ELGs, are generally comprised of five
polypeptide subunits, Unwin, N. (1993), Cell 72: 31-41; Unwin, N. (1995),
Nature
373: 37-43; Hucho, F., et al., (1996) J. Neurochem. 66: 1781-1792; Hucho, F.,
et al.,
(1996) Eur. J. Biochem. 239: 539-557; Alexander, S.P.H. and J.A. Peters
(1997),
Trends Pharmacol. Sci., Elsevier, pp. 4-6; 36-40; 42-44; and Xue, H. (1998) J.
Mol.
Evol. 47: 323-333. Each subunit has 4 membrane spanning regions: this serves
as a
means of identifying other members of the ELG family of proteins. ELG bind a
ligand and in response modulate the flow of ions. Examples of ELG include most
members of the neurotransmitter-receptor family of proteins, e.g., GABAI
receptors.
Other members of this family of ion channels include glycine receptors,
ryandyne
receptors, and ligand gated calcium channels.
The Voltage-gated Ion Channel (VIC) Superfamily
Proteins of the VIC family are ion-selective charnel proteins found in a wide
range of bacteria, archaea and eukaryotes Hille, B. (1992), Chapter 9:
Structure of
channel proteins; Chapter 20: Evolution and diversity. In: Ionic Channels of
Excitable
Membranes, 2nd Ed., Sinaur Assoc. Inc., Pubs., Sunderland, Massachusetts;
Sigworth, F.J. (1993), Quart. Rev. Biophys. 27: 1-40; Salkoff, L. and T. Jegla
(1995),
Neuron 15: 489-492; Alexander, S.P.H. et al., (1997), Trends Pharmacol. Sci.,
Elsevier, pp. 76-84; Jan, L.Y. et al., (1997), Annu. Rev. Neurosci. 20: 91-
123; Doyle,
D.A, et al., (1998) Science 280: 69-77; Terlau, H. and W. Stiihmer (1998),
Naturwissenschaften 85: 437-444. They are often homo- or heterooligomeric
structures with several dissimilar subunits (e.g., al-a2-d-b Ca2+ channels,
ab~bz Na+
channels or (a)4-b K+ channels), but the channel and the primary receptor is
usually
associated with the a (or al) subunit. Functionally characterized members are
specific
for K+, Na+ or Ca2+. The K+ channels usually consist of homotetrameric
structures
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with each a-subunit possessing six transmembrane spanners (TMSs). The al and a
subunits of the Ca2+ and Na+ channels, respectively, are about four times as
large and
possess 4 units, each with 6 TMSs separated by a hydrophilic loop, for a total
of 24
TMSs. These large channel proteins form heterotetra-unit structures equivalent
to the
homotetrameric structures of most K+ channels. All four units of the Ca2+ and
Na+
channels are homologous to the single unit in the homotetrameric K+ channels.
Ion
flux via the eukaryotic channels is generally controlled by the transmembrane
electrical potential (hence the designation, voltage-sensitive) although some
are
controlled by ligand or receptor binding.
Several putative K+-selective channel proteins of the VIC family have been
identified in prokaryotes. The structure of one of them, the KcsA K+ channel
of
Streptomyces lividans, has been solved to 3.2 ~ resolution. The protein
possesses four
identical subunits, each with two transmembrane helices, arranged in the shape
of an
inverted teepee or cone. The cone cradles the "selectivity filter" P domain in
its outer
end. The narrow selectivity filter is only 12 ~ long, whereas the remainder of
the
channel is wider and lined with hydrophobic residues. A large water-filled
cavity and
helix dipoles stabilize K+ in the pore. The selectivity filter has two bound
K+ ions
about 7.5 A apart from each other. Ion conduction is proposed to result from a
balance
of electrostatic attractive and repulsive forces.
In eukaryotes, each VIC family channel type has several subtypes based on
pharmacological and electrophysiological data. Thus, there are five types of
Caz+
channels (L, N, P, Q and T). There are at least ten types of K+ channels, each
responding in different ways to different stimuli: voltage-sensitive [Ka, Kv,
Kvr, Kvs
and Ksr], Ca2+-sensitive [BK~a, IK~a and SKCa] and receptor-coupled [KM and
KACn].
There are at least six types of Na+ channels (I, II, III, p1, H1 and PN3).
Tetrameric
channels from both prokaryotic and eukaryotic organisms are known in which
each a-
subunit possesses 2 TMSs rather than 6, and these two TMSs are homologous to
TMSs 5 and 6 of the six TMS unit found in the voltage-sensitive channel
proteins.
KcsA of S. lividans is an example of such a 2 TMS channel protein. These
channels
may include the KNa (Na+-activated) and Kvo, (cell volume-sensitive) K+
channels, as
well as distantly related channels such as the Tokl K+ channel of yeast, the
TWIK-1
inward rectifier K+ channel of the mouse and the TREK-1 K+ channel of the
mouse.
Because of insufficient sequence similarity with proteins of the VIC family,
inward
rectifier K+ IRK channels (ATP-regulated; G-protein-activated) which possess a
P
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domain and two flanking TMSs are placed in a distinct family. However,
substantial
sequence similarity in the P region suggests that they are homologous. The b,
g and d
subunits of VIC family members, when present, frequently play regulatory roles
in
channel activation/deactivation.
The Epithelial Na+ Channel (ENaC Family
The ENaC family consists of over twenty-four sequenced proteins (Canessa,
C.M., et al., (1994), Nature 367: 463-467, Le, T. and M.H. Saier, Jr. (1996),
Mol.
Membr. Biol. 13: 149-157; Garty, H. and L.G. Palmer (1997), Physiol. Rev. 77:
359-
396; Waldmann, R., et al., (1997), Nature 386: 173-177; Darboux, L, et al.,
(1998), J.
Biol. Chem. 273: 9424-9429; Firsov, D., et al., (1998), EMBO J. 17: 344-352;
Horisberger, J.-D. (1998). Curr. Opin. Struc. Biol. 10: 443-449). All are from
animals
with no recognizable homologues in other eukaryotes or bacteria. The
vertebrate
ENaC proteins from epithelial cells cluster tightly together on the
phylogenetic tree:
voltage-insensitive ENaC homologues are also found in the brain. Eleven
sequenced
C. elegans proteins, including the degenerins, are distantly related to the
vertebrate
proteins as well as to each other. At least some of these proteins form part
of a
mechano-transducing complex for touch sensitivity. The homologous Helix
aspersa
(FMRF-amide)-activated Na+ channel is the first peptide neurotransmitter-gated
ionotropic receptor to be sequenced.
Protein members of this family all exhibit the same apparent topology, each
with N- and C-termini on the inside of the cell, two amphipathic transmembrane
spanning segments, and a large extracellular loop. The extracellular domains
contain
numerous highly conserved cysteine residues. They are proposed to serve a
receptor
function.
Mammalian ENaC is important for the maintenance of Na+ balance and the
regulation of blood pressure. Three homologous ENaC subunits, alpha, beta, and
gamma, have been shown to assemble to form the highly Na +-selective channel.
The
stoichiometry of the three subunits is alpha2, betal, gammal in a
heterotetrameric
architecture.
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The Glutamate-gated Ion Channel (GIC) Famil~of Neurotransmitter
Receptors
Members of the GIC family are heteropentameric complexes in which each of
the 5 subunits is of 800-1000 amino acyl residues in length (Nakanishi, N., et
al,
(1990), Neuron 5: 569-581; Unwin, N. (1993), Cell 72: 31-41; Alexander, S.P.H.
and
J.A. Peters (1997) Trends Pharmacol. Sci., Elsevier, pp. 36-40). These
subunits may
span the membrane three or five times as putative a-helices with the N-termini
(the
glutamate-binding domains) localized extracellularly and the C-termini
localized
cytoplasmically. They may be distantly related to the ligand-gated ion
channels, and if
so, they may possess substantial b-structure in their transmembrane regions.
However,
homology between these two families cannot be established on the basis of
sequence
comparisons alone. The subunits fall into six subfamilies: a, b, g, d, a and
z.
The GIC channels are divided into three types: (1) a-amino-3-hydroxy-5-
methyl-4-isoxazole propionate (AMPA)-, (2) kainate- and (3) N-methyl-D-
aspartate
(NMDA)-selective glutamate receptors. Subunits of the AMPA and kainate classes
exhibit 35-40% identity with each other while subunits of the NMDA receptors
exhibit 22-24% identity with the former subunits. They possess large N-
terminal,
extracellular glutamate-binding domains that are homologous to the periplasmic
glutamine and glutamate receptors of ABC-type uptake permeases of Gram-
negative
bacteria. All known members of the GIC family are from animals. The different
channel (receptor) types exhibit distinct ion selectivities and conductance
properties.
The NMDA-selective large conductance channels are highly permeable to
monovalent cations and Ca2+. The AMPA- and kainate-selective ion channels are
permeable primarily to monovalent cations with only low permeability to Ca2+.
The Chloride Channel~ClC) Family
The C1C family is a large family consisting of dozens of sequenced proteins
derived from Gram-negative and Gram-positive bacteria, cyanobacteria, archaea,
yeast, plants and animals (Steinmeyer, K., et al., (1991), Nature 354: 301-
304;
Uchida, S., et al., (1993), J. Biol. Chem. 268: 3821-3824; Huang, M.-E., et
al., (1994),
J. Mol. Biol. 242: 595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604;
Fisher,
W.E., et al., (1995), Genomics. 29:598-606; and Foskett, J.K. (1998), Annu.
Rev.
Physiol. 60: 689-717). These proteins are essentially ubiquitous, although
they are not
encoded within genomes of Haemophilus influenzae, Mycoplasma genitalium, and
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Mycoplasma pneumoniae. Sequenced proteins vary in size from 395 amino acyl
residues (M. jannaschii) to 988 residues (man). Several organisms contain
multiple
C1C family paralogues. For example, Synechocystis has two paralogues, one of
451
residues in length and the other of 899 residues. Arabidopsis thaliana has at
least four
sequenced paralogues, (775-792 residues), humans also have at least five
paralogues
(820-988 residues), and C. elegans also has at least five (810-950 residues).
There are
nine known members in mammals, and mutations in three of the corresponding
genes
cause human diseases. E. coli, Methanococcusjannaschii and Saccharomyces
cerevisiae only have one C1C family member each. With the exception of the
larger
Synechocystis paralogue, all bacterial proteins are small (395-492 residues)
while all
eukaryotic proteins are larger (687-988 residues). These proteins exhibit 10-
12
putative transmembrane a-helical spanners (TMSs) and appear to be present in
the
membrane as homodimers. While one member of the family, Torpedo C1C-O, has
been reported to have two channels, one per subunit, others are believed to
have just
one.
All functionally characterized members of the CIC family transport chloride,
some in a voltage-regulated process. These channels serve a variety of
physiological
functions (cell volume regulation; membrane potential stabilization; signal
transduction; transepithelial transport, etc.). Different homologues in humans
exhibit
differing anion selectivities, i.e., C1C4 and C1C5 share a N03' > Cf > Br > I'
conductance sequence, while C1C3 has an I' > CI' selectivity. The C1C4 and
C1C5
channels and others exhibit outward rectifying currents with currents only at
voltages
more positive than +20mV.
Animal Inward Rectifier K+ Channel (IRK-C) Familx
IRK channels possess the "minimal channel-forming structure" with only a P
domain, characteristic of the channel proteins of the VIC family, and two
flanking
transmembrane spanners (Shuck, M.E., et al., (1994), J. Biol. Chem. 269: 24261-
24270; Ashen, M.D., et al., (1995), Am. J. Physiol. 268: H506-H511; Salkoff,
L. and
T. Jegla (1995), Neuron 15: 489-492; Aguilar-Bryan, L., et al., (1998),
Physiol. Rev.
78: 227-245; Ruknudin, A., et al., (1998), J. Biol. Chem. 273: 14165-14171).
They
may exist in the membrane as homo- or heterooligomers. They have a greater
tendency to let K+ flow into the cell than out. Voltage-dependence may be
regulated
by external K+, by internal Mg2+, by internal ATP and/or by G-proteins. The P
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domains of IRK channels exhibit limited sequence similarity to those of the
VIC
family, but this sequence similarity is insufficient to establish homology.
Inward
rectifiers play a role in setting cellular membrane potentials, and the
closing of these
channels upon depolarization permits the occurrence of long duration action
potentials
5 with a plateau phase. Inward rectifiers lack the intrinsic voltage sensing
helices found
in VIC family channels. In a few cases, those of Kirl.la and Kir6.2, for
example,
direct interaction with a member of the ABC superfamily has been proposed to
confer
unique functional and regulatory properties to the heteromeric complex,
including
sensitivity to ATP. The SUR1 sulfonylurea receptor (spQ09428) is the ABC
protein
10 that regulates the Kir6.2 channel in response to ATP, and CFTR may regulate
Kirl.la. Mutations in SUR1 are the cause of familial persistent
hyperinsulinemic
hypoglycemia in infancy (PHHI), an autosomal recessive disorder characterized
by
unregulated insulin secretion in the pancreas.
ATP dated Cation Channel (ACC) Family
Members of the ACC family (also called P2X receptors) respond to ATP, a
functional neurotransmitter released by exocytosis from many types of neurons
(North, R.A. (1996), Curr. Opin. Cell Biol. 8: 474-483; Soto, F., M. Garcia-
Guzman
and W. Stiihmer (1997), J. Membr. Biol. 160: 91-100). They have been placed
into
seven groups (P2X~ - P2X~) based on their pharmacological properties. These
channels, which function at neuron-neuron and neuron-smooth muscle junctions,
may
play roles in the control of blood pressure and pain sensation. They may also
function
in lymphocyte and platelet physiology. They are found only in animals.
The proteins of the ACC family are quite similar in sequence (>35% identity),
but they possess 380-1000 amino acyl residues per subunit with variability in
length
localized primarily to the C-terminal domains. They possess two transmembrane
spanners, one about 30-50 residues from their N-termini, the other near
residues 320-
340. The extracellular receptor domains between these two spanners (of about
270
residues) are well conserved with numerous conserved glycyl and cysteyl
residues.
The hydrophilic C-termini vary in length from 25 to 240 residues. They
resemble the
topologically similar epithelial Na+ channel (ENaC) proteins in possessing (a)
N- and
C-termini localized intracellularly, (b) two putative transmembrane spanners,
(c) a
large extracellular loop domain, and (d) many conserved extracellular cysteyl
residues. ACC family members are, however, not demonstrably homologous with
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them. ACC channels are probably hetero- or homomultimers and transport small
monovalent canons (Me+). Some also transport Ca2+; a few also transport small
metabolites.
The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca2+ Channel (RIR-CaC)
Family
Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate (IP3)-sensitive Ca2+-
release channels function in the release of Ca2+ from intracellular storage
sites in
animal cells and thereby regulate various Caz+ -dependent physiological
processes
(Hasan, G. et al., (1992) Development 116: 967-975; Michikawa, T., et al.,
(1994), J.
Biol. Chem. 269: 9184-9189; Tunwell, R.E.A., (1996), Biochem. J. 318: 477-487;
Lee, A.G. (1996) Biomembranes, Vol. 6, Transmembrane Receptors and Channels
(A.G. Lee, ed.), JAI Press, Denver, CO., pp 291-326; Mikoshiba, K., et al.,
(1996) J.
Biochem. Biomem. 6: 273-289). Ry receptors occur primarily in muscle cell
sarcoplasmic reticular (SR) membranes, and IP3 receptors occur primarily in
brain
cell endoplasmic reticular (ER) membranes where they effect release of Caz+
into the
cytoplasm upon activation (opening) of the channel.
The Ry receptors are activated as a result of the activity of dihydropyridine-
sensitive Caz+ channels. The latter are members of the voltage-sensitive ion
channel
(VIC) family. Dihydropyridine-sensitive channels are present in the T-tubular
systems
of muscle tissues.
Ry receptors are homotetrameric complexes with each subunit exhibiting a
molecular size of over 500,000 daltons (about 5,000 amino acyl residues). They
possess C-terminal domains with six putative transmembrane a -helical spanners
(TMSs). Putative pore-forming sequences occur between the fifth and sixth TMSs
as
suggested for members of the VIC family. The large N-terminal hydrophilic
domains
and the small C-terminal hydrophilic domains are localized to the cytoplasm.
Low
resolution 3-dimensional structural data are available. Mammals possess at
least three
isoforms that probably arose by gene duplication and divergence before
divergence of
the mammalian species. Homologues are present in humans and Caenorabditis
elegans.
IP3 receptors resemble Ry receptors in many respects. (1) They are
homotetrameric complexes with each subunit exhibiting a molecular size of over
300,000 daltons (about 2,700 amino acyl residues). (2) They possess C-terminal
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channel domains that are homologous to those of the Ry receptors. (3) The
channel
domains possess six putative TMSs and a putative channel lining region between
TMSs 5 and 6. (4) Both the large N-terminal domains and the smaller C-terminal
tails
face the cytoplasm. (5) They possess covalently linked carbohydrate on
extracytoplasmic loops of the channel domains. (6) They have three currently
recognized isoforms (types 1, 2, and 3) in mammals which are subject to
differential
regulation and have different tissue distributions.
IP3 receptors possess three domains: N-terminal IP3-binding domains, central
coupling or regulatory domains and C-terminal channel domains. Channels are
activated by IP3 binding, and like the Ry receptors, the activities of the IP3
receptor
channels are regulated by phosphorylation of the regulatory domains, catalyzed
by
various protein kinases. They predominate in the endoplasmic reticular
membranes of
various cell types in the brain but have also been found in the plasma
membranes of
some nerve cells derived from a variety of tissues.
The channel domains of the Ry and IP3 receptors comprise a coherent family
that in spite of apparent structural similarities, do not show appreciable
sequence
similarity of the proteins of the VIC family. The Ry receptors and the IP3
receptors
cluster separately on the RIR-CaC family tree. They both have homologues in
Drosophila. Based on the phylogenetic tree for the family, the family probably
evolved in the following sequence: (1) A gene duplication event occurred that
gave
rise to Ry and IP3 receptors in invertebrates. (2) Vertebrates evolved from
invertebrates. (3) The three isoforms of each receptor arose as a result of
two distinct
gene duplication events. (4) These isoforms were transmitted to mammals before
divergence of the mammalian species.
The Organellar Chloride Channel (O-C1C) Family
Proteins of the O-C1C family are voltage-sensitive chloride channels found in
intracellular membranes but not the plasma membranes of animal cells (Landry,
D, et
al., (1993), J. Biol. Chem. 268: 14948-14955; Valenzuela, Set al., (1997), J.
Biol.
Chem. 272: 12575-12582; and Duncan, R.R., et al., (1997), J. Biol. Chem. 272:
23880-23886).
They are found in human nuclear membranes, and the bovine protein targets to
the microsomes, but not the plasma membrane, when expressed in Xenopus laevis
oocytes. These proteins are thought to function in the regulation of the
membrane
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13
potential and in transepithelial ion absorption and secretion in the kidney.
They
possess two putative transmembrane a-helical spanners (TMSs) with cytoplasmic
N-
and C-termini and a large luminal loop that may be glycosylated. The bovine
protein
is 437 amino acyl residues in length and has the two putative TMSs at
positions 223-
239 and 367-385. The human nuclear protein is much smaller (241 residues). A
C.
elegans homologue is 260 residues long.
Sodium/Chloride-Dependent Organic Solute Cotransporter
The novel human protein, and encoding gene, provided by the present invention
is related to the family of sodium/chloride-dependent organic solute
cotransporters,
which includes cotransporters for neurotransmitters, amino acids, and organic
osmolytes. A new member of the family, ROSIT (renal osmotic stress-induced
transporter) has recently been identified (Wasserman et al., Am JPhysiol 1994
Oct;267(4 Pt 2):F688-94). Genes for sodium/chloride-dependent organic solute
cotransporters are known to undergo extensive alternative splicing in order to
generate
numerous isoforms. For example, the XT2 gene generates six different isoforms
through
alternative splicing (Hash et al., Receptors Channels 1998;6(2):113-28).
The sodium:neurotransmitter symporters (SNF) are a subfamily of
sodium/chloride-dependent organic solute cotransporters. These transporter
proteins are
important for sodium-dependent reuptake of a wide variety of neurotransmitters
into
presynaptic terminals, which ceases neurotransmitter action. SNF transporters
are targets
for psychomotor stimulants such as amphetamines and cocaine, which prevent
neurotransmitter reuptake by SNFs, thereby prolonging neurotransmitter action.
Transporter proteins, particularly members of the sodium/chloride-dependent
organic solute cotransporter subfamily, are a major target for drug action and
development. Accordingly, it is valuable to the field of pharmaceutical
development to
identify and characterize previously unknown transport proteins. The present
invention
advances the state of the art by providing previously unidentified human
transport
proteins.
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SUMMARY OF THE INVENTION
The present invention is based in part on the identification of amino acid
sequences of human transporter peptides and proteins that are related to the
sodium/chloride-dependent organic solute cotransporter subfamily, as well as
allelic
variants and other mammalian orthologs thereof. These unique peptide
sequences,
and nucleic acid sequences that encode these peptides, can be used as models
for the
development of human therapeutic targets, aid in the identification of
therapeutic
proteins, and serve as targets for the development of human therapeutic agents
that
modulate transporter activity in cells and tissues that express the
transporter.
Experimental data as provided in Figure 1 indicates expression in humans in
the
colon, kidney, and neuron tumors.
DESCRIPTION OF THE FIGURE SHEETS
FIGURE 1 provides the nucleotide sequence of a cDNA molecule that
encodes the transporter protein of the present invention. (SEQ ID NO:1) In
addition
structure and functional information is provided, such as ATG start, stop and
tissue
distribution, where available, that allows one to readily determine specific
uses of
inventions based on this molecular sequence. Experimental data as provided in
Figure 1 indicates expression in humans in the colon, kidney, and neuron
tumors.
FIGURE 2 provides the predicted amino acid sequence of the transporter of
the present invention. (SEQ ID N0:2) In addition structure and functional
information such as protein family, function, and modification sites is
provided where
available, allowing one to readily determine specific uses of inventions based
on this
molecular sequence.
FIGURE 3 provides genomic sequences that span the gene encoding the
transporter protein of the present invention. (SEQ ID N0:3) As illustrated in
Figure
3, SNPs were identified at 23 different nucleotide positions.
DETAILED DESCRIPTION OF THE INVENTION
General Description
The present invention is based on the sequencing of the human genome.
During the sequencing and assembly of the human genome, analysis of the
sequence
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information revealed previously unidentified fragments of the human genome
that
encode peptides that share structural and/or sequence homology to
protein/peptide/domains identified and characterized within the art as being a
transporter protein or part of a transporter protein and are related to the
5 sodium/chloride-dependent organic solute cotransporter subfamily. Utilizing
these
sequences, additional genomic sequences were assembled and transcript and/or
cDNA
sequences were isolated and characterized. Based on this analysis, the present
invention provides amino acid sequences of human transporter peptides and
proteins
that are related to the sodium/chloride-dependent organic solute cotransporter
10 subfamily, nucleic acid sequences in the form of transcript sequences, cDNA
sequences and/or genomic sequences that encode these transporter peptides and
proteins, nucleic acid variation (allelic information), tissue distribution of
expression,
and information about the closest art known protein/peptide/domain that has
structural
or sequence homology to the transporter of the present invention.
15 In addition to being previously unknown, the peptides that are provided in
the
present invention are selected based on their ability to be used for the
development of
commercially important products and services. Specifically, the present
peptides are
selected based on homology and/or structural relatedness to known transporter
proteins of the sodium/chloride-dependent organic solute cotransporter
subfamily and
the expression pattern observed. Experimental data as provided in Figure 1
indicates
expression in humans in the colon, kidney, and neuron tumors.. The art has
clearly
established the commercial importance of members of this family of proteins
and
proteins that have expression patterns similar to that of the present gene.
Some of the
more specific features of the peptides of the present invention, and the uses
thereof,
are described herein, particularly in the Background of the Invention and in
the
annotation provided in the Figures, and/or are known within the art for each
of the
known sodium/chloride-dependent organic solute cotransporter family or
subfamily of
transporter proteins.
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16
Specific Embodiments
Peptide Molecules
The present invention provides nucleic acid sequences that encode protein
molecules that have been identified as being members of the transporter family
of
proteins and are related to the sodium/chloride-dependent organic solute
cotransporter
subfamily (protein sequences are provided in Figure 2, transcript/cDNA
sequences are
provided in Figures l and genomic sequences are provided in Figure 3). The
peptide
sequences provided in Figure 2, as well as the obvious variants described
herein,
particularly allelic variants as identified herein and using the information
in Figure 3,
will be referred herein as the transporter peptides of the present invention,
transporter
peptides, or peptides/proteins of the present invention.
The present invention provides isolated peptide and protein molecules that
consist of, consist essentially of, or comprising the amino acid sequences of
the
transporter peptides disclosed in the Figure 2, (encoded by the nucleic acid
molecule
shown in Figure 1, transcript/cDNA or Figure 3, genomic sequence), as well as
all
obvious variants of these peptides that are within the art to make and use.
Some of
these variants are described in detail below.
As used herein, a peptide is said to be "isolated" or "purified" when it is
substantially free of cellular material or free of chemical precursors or
other
chemicals. The peptides of the present invention can be purified to
homogeneity or other
degrees of purity. The level of purification will be based on the intended
use. The
critical feature is that the preparation allows for the desired function of
the peptide, even
if in the presence of considerable amounts of other components (the features
of an
isolated nucleic acid molecule is discussed below).
In some uses, "substantially free of cellular material" includes preparations
of the
peptide having less than about 30% (by dry weight) other proteins (i.e.,
contaminating
protein), less than about 20% other proteins, less than about 10% other
proteins, or less
than about S% other proteins. When the peptide is recombinantly produced, it
can also
be substantially free of culture medium, i.e., culture medium represents less
than about
20% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of the peptide in which it is separated from chemical
precursors or
other chemicals that are involved in its synthesis. In one embodiment, the
language
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17
"substantially free of chemical precursors or other chemicals" includes
preparations of
the transporter peptide having less than about 30% (by dry weight) chemical
precursors
or other chemicals, less than about 20% chemical precursors or other
chemicals, less
than about 10% chemical precursors or other chemicals, or less than about 5%
chemical
precursors or other chemicals.
The isolated transporter peptide can be purified from cells that naturally
express
it, purified from cells that have been altered to express it (recombinant), or
synthesized
using known protein synthesis methods. Experimental data as provided in Figure
I
indicates expression in humans in the colon, kidney, and neuron tumors. For
example, a
nucleic acid molecule encoding the transporter peptide is cloned into an
expression
vector, the expression vector introduced into a host cell and the protein
expressed in the
host cell. The protein can then be isolated from the cells by an appropriate
purification
scheme using standard protein purification techniques. Many of these
techniques are
described in detail below.
Accordingly, the present invention provides proteins that consist of the amino
acid sequences provided in Figure 2 (SEQ ID N0:2), for example, proteins
encoded by
the transcript/cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NO:1 )
and the
genomic sequences provided in Figure 3 (SEQ ID N0:3). The amino acid sequence
of
such a protein is provided in Figure 2. A protein consists of an amino acid
sequence
when the amino acid sequence is the final amino acid sequence of the protein.
The present invention further provides proteins that consist essentially of
the
amino acid sequences provided in Figure 2 (SEQ ID N0:2), for example, proteins
encoded by the transcript/cDNA nucleic acid sequences shown in Figure 1 (SEQ
ID
NO:1) and the genomic sequences provided in Figure 3 (SEQ ID N0:3). A protein
consists essentially of an amino acid sequence when such an amino acid
sequence is
present with only a few additional amino acid residues, for example from about
1 to
about 100 or so additional residues, typically from 1 to about 20 additional
residues in
the final protein.
The present invention further provides proteins that comprise the amino acid
sequences provided in Figure 2 (SEQ ID N0:2), for example, proteins encoded by
the
transcript/cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NO:1 ) and
the
genomic sequences provided in Figure 3 (SEQ ID N0:3). A protein comprises an
amino
acid sequence when the amino acid sequence is at least part of the final amino
acid
sequence of the protein. In such a fashion, the protein can be only the
peptide or have
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18
additional amino acid molecules, such as amino acid residues (contiguous
encoded
sequence) that are naturally associated with it or heterologous amino acid
residues/peptide sequences. Such a protein can have a few additional amino
acid
residues or can comprise several hundred or more additional amino acids. The
preferred
classes of proteins that are comprised of the transporter peptides of the
present invention
are the naturally occurring mature proteins. A brief description of how
various types of
these proteins can be made/isolated is provided below.
The transporter peptides of the present invention can be attached to
heterologous
sequences to form chimeric or fusion proteins. Such chimeric and fusion
proteins
comprise a transporter peptide operatively linked to a heterologous protein
having an
amino acid sequence not substantially homologous to the transporter peptide.
"Operatively linked" indicates that the transporter peptide and the
heterologous protein
are fused in-frame. The heterologous protein can be fused to the N-terminus or
C-
terminus of the transporter peptide.
In some uses, the fusion protein does not affect the activity of the
transporter
peptide per se. For example, the fusion protein can include, but is not
limited to,
enzymatic fusion proteins, for example beta-galactosidase fixsions, yeast two-
hybrid
GAL fizsions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such
fusion
proteins, particularly poly-His fi~sions, can facilitate the purification of
recombinant
transporter peptide. In certain host cells (e.g., mammalian host cells),
expression and/or
secretion of a protein can be increased by using a heterologous signal
sequence.
A chimeric or fusion protein can be produced by standard recombinant DNA
techniques. For example, DNA fragments coding for the different protein
sequences are
ligated together in-frame in accordance with conventional techniques. In
another
embodiment, the fusion gene can be synthesized by conventional techniques
including
automated DNA synthesizers. Alternatively, PCR amplification of gene fragments
can
be carried out using anchor primers which give rise to complementary overhangs
between two consecutive gene fragments which can subsequently be annealed and
re-
amplified to generate a chimeric gene sequence (see Ausubel et al., Current
Protocols in
Molecular Biology, 1992). Moreover, many expression vectors are commercially
available that already encode a fusion moiety (e.g., a GST protein). A
transporter
peptide-encoding nucleic acid can be cloned into such an expression vector
such that the
fusion moiety is linked in-frame to the transporter peptide.
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19
As mentioned above, the present invention also provides and enables obvious
variants of the amino acid sequence of the proteins of the present invention,
such as
naturally occurnng mature forms of the peptide, allelic/sequence variants of
the peptides,
non-naturally occurnng recombinantly derived variants of the peptides, and
orthologs
and paralogs of the peptides. Such variants can readily be generated using art-
known
techniques in the fields of recombinant nucleic acid technology and protein
biochemistry. It is understood, however, that variants exclude any amino acid
sequences
disclosed prior to the invention.
Such variants can readily be identified/made using molecular techniques and
the
sequence information disclosed herein. Further, such variants can readily be
distinguished from other peptides based on sequence and/or structural homology
to the
transporter peptides of the present invention. The degree of homology/identity
present
will be based primarily on whether the peptide is a functional variant or non-
functional
variant, the amount of divergence present in the paralog family and the
evolutionary
distance between the orthologs.
To determine the percent identity of two amino acid sequences or two nucleic
acid sequences, the sequences are aligned for optimal comparison purposes
(e.g., gaps
can be introduced in one or both of a first and a second amino acid or nucleic
acid
sequence for optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%,
60%,
70%, 80%, or 90% or more of a reference sequence is aligned for comparison
purposes. The amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a position in the
first
sequence is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules are
identical at that
position (as used herein amino acid or nucleic acid "identity" is equivalent
to amino
acid or nucleic acid "homology"). The percent identity between the two
sequences is
a function of the number of identical positions shared by the sequences,
taking into
account the number of gaps, and the length of each gap, which need to be
introduced
for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity and
similarity between two sequences can be accomplished using a mathematical
algorithm. (Computational Molecular Biology, Lesk, A.M., ed., Oxford
University
Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith,
D.W.,
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ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part
I ,
Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994;
Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and
Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New
York,
5 1991). In a preferred embodiment, the percent identity between two amino
acid
sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-
453 (1970)) algorithm which has been incorporated into the GAP program in the
GCG software package (available at http://www.gcg.com), using either a Blossom
62
matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and
a length
10 weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the
percent identity
between two nucleotide sequences is determined using the GAP program in the
GCG
software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984))
(available
at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40,
50,
60, 70, or 80 and a length weight of l, 2, 3, 4, 5, or 6. In another
embodiment, the
15 percent identity between two amino acid or nucleotide sequences is
determined using
the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has
been
incorporated into the ALIGN program (version 2.0), using a PAM120 weight
residue
table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences of the present invention can further be
20 used as a "query sequence" to perform a search against sequence databases
to, for
example, identify other family members or related sequences. Such searches can
be
performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be
performed
with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide
sequences homologous to the nucleic acid molecules of the invention. BLAST
protein searches can be performed with the XBLAST program, score = 50,
wordlength = 3 to obtain amino acid sequences homologous to the proteins of
the
invention. To obtain gapped alignments for comparison purposes, Gapped BLAST
can be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402
(1997)). When utilizing BLAST and gapped BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
Full-length pre-processed forms, as well as mature processed forms, of
proteins
that comprise one of the peptides of the present invention can readily be
identified as
having complete sequence identity to one of the transporter peptides of the
present
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21
invention as well as being encoded by the same genetic locus as the
transporter peptide
provided herein. The gene encoding the novel transporter protein of the
present
invention is located on human chromosome 5.
Allelic variants of a transporter peptide can readily be identified as being a
S human protein having a high degree (significant) of sequence
homology/identity to at
least a portion of the transporter peptide as well as being encoded by the
same genetic
locus as the transporter peptide provided herein. Genetic locus can readily be
determined based on the genomic information provided in Figure 3, such as the
genomic
sequence mapped to the reference human. The gene encoding the novel
transporter
protein of the present invention is located on human chromosome 5. As used
herein,
two proteins (or a region of the proteins) have significant homology when the
amino
acid sequences are typically at least about 70-80%, 80-90%, and more typically
at
least about 90-95% or more homologous. A significantly homologous amino acid
sequence, according to the present invention, will be encoded by a nucleic
acid
sequence that will hybridize to a transporter peptide encoding nucleic acid
molecule
under stringent conditions as more fully described below.
Figure 3 provides information on SNPs that have been found in the gene
encoding the transporter protein of the present invention. SNPs were
identified at 23
different nucleotide positions. Changes in the amino acid sequence caused by
these
SNPs can readily be determined using the universal genetic code and the
protein
sequence provided in Figure 2 as a reference. These SNPs may also affect
control/regulatory elements.
Paralogs of a transporter peptide can readily be identified as having some
degree
of significant sequence homology/identity to at least a portion of the
transporter peptide,
as being encoded by a gene from humans, and as having similar activity or
function.
Two proteins will typically be considered paralogs when the amino acid
sequences are
typically at least about 60% or greater, and more typically at least about 70%
or
greater homology through a given region or domain. Such paralogs will be
encoded
by a nucleic acid sequence that will hybridize to a transporter peptide
encoding
nucleic acid molecule under moderate to stringent conditions as more fully
described
below.
Orthologs of a transporter peptide can readily be identified as having some
degree of significant sequence homology/identity to at least a portion of the
transporter
peptide as well as being encoded by a gene from another organism. Preferred
orthologs
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22
will be isolated from mammals, preferably primates, for the development of
human
therapeutic targets and agents. Such orthologs will be encoded by a nucleic
acid
sequence that will hybridize to a transporter peptide encoding nucleic acid
molecule
under moderate to stringent conditions, as more fully described below,
depending on
the degree of relatedness of the two organisms yielding the proteins.
Non-naturally occurnng variants of the transporter peptides of the present
invention can readily be generated using recombinant techniques. Such variants
include,
but are not limited to deletions, additions and substitutions in the amino
acid sequence of
the transporter peptide. For example, one class of substitutions are conserved
amino
acid substitution. Such substitutions are those that substitute a given amino
acid in a
transporter peptide by another amino acid of like characteristics. Typically
seen as
conservative substitutions are the replacements, one for another, among the
aliphatic
amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser
and Thr;
exchange of the acidic residues Asp and Glu; substitution between the amide
residues
Asn and Gln; exchange of the basic residues Lys and Arg; and replacements
among the
aromatic residues Phe and Tyr. Guidance concerning which amino acid changes
are
likely to be phenotypically silent are found in Bowie et al., Science 247:1306-
1310
( 1990).
Variant transporter peptides can be fully functional or can lack function in
one or
more activities, e.g. ability to bind ligand, ability to transport ligand,
ability to mediate
signaling, etc. Fully functional variants typically contain only conservative
variation or
variation in non-critical residues or in non-critical regions. Figure 2
provides the result
of protein analysis and can be used to identify critical domains/regions.
Functional
variants can also contain substitution of similar amino acids that result in
no change or
an insignificant change in function. Alternatively, such substitutions may
positively or
negatively affect function to some degree.
Non-functional variants typically contain one or more non-conservative amino
acid substitutions, deletions, insertions, inversions, or truncation or a
substitution,
insertion, inversion, or deletion in a critical residue or critical region.
Amino acids that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham et al., Science 244:1081-1085 (1989)), particularly using the
results
provided in Figure 2. The latter procedure introduces single alanine mutations
at every
residue in the molecule. The resulting mutant molecules are then tested for
biological
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23
activity such as transporter activity or in assays such as an in vitro
proliferative activity.
Sites that are critical for binding partner/substrate binding can also be
determined by
structural analysis such as crystallization, nuclear magnetic resonance or
photoaffmity
labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.
Science 255:306-
312 ( 1992)).
The present invention further provides fragments of the transporter peptides,
in
addition to proteins and peptides that comprise and consist of such fragments,
particularly those comprising the residues identified in Figure 2. The
fragments to which
the invention pertains, however, are not to be construed as encompassing
fragments that
may be disclosed publicly prior to the present invention.
As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more
contiguous amino acid residues from a transporter peptide. Such fragments can
be
chosen based on the ability to retain one or more of the biological activities
of the
transporter peptide or could be chosen for the ability to perform a function,
e.g. bind a
substrate or act as an immunogen. Particularly important fragments are
biologically
active fragments, peptides that are, for example, about 8 or more amino acids
in length.
Such fragments will typically comprise a domain or motif of the transporter
peptide, e.g.,
active site, a transmembrane domain or a substrate-binding domain. Further,
possible
fragments include, but are not limited to, domain or motif containing
fragments, soluble
peptide fragments, and fragments containing immunogenic structures. Predicted
domains
and functional sites are readily identifiable by computer programs well known
and
readily available to those of skill in the art (e.g., PROSITE analysis). The
results of one
such analysis are provided in Figure 2.
Polypeptides often contain amino acids other than the 20 amino acids commonly
referred to as the 20 naturally occurnng amino acids. Further, many amino
acids,
including the terminal amino acids, may be modified by natural processes, such
as
processing and other post-translational modifications, or by chemical
modification
techniques well known in the art. Common modifications that occur naturally in
transporter peptides are described in basic texts, detailed monographs, and
the research
literature, and they are well known to those of skill in the art (some of
these features are
identified in Figure 2).
Known modifications include, but are not limited to, acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin, covalent
attachment of a
heme moiety, covalent attachment of a nucleotide or nucleotide derivative,
covalent
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24
attachment of a lipid or lipid derivative, covalent attachment of
phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation, demethylation, formation
of
covalent crosslinks, formation of cystine, formation of pyroglutamate,
formylation,
gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination,
S methylation, myristoylation, oxidation, proteolytic processing,
phosphorylation,
prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated
addition of
amino acids to proteins such as arginylation, and ubiquitination.
Such modifications are well known to those of skill in the art and have been
described in great detail in the scientific literature. Several particularly
common
modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation
of
glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are
described
in most basic texts, such as Proteins - Structure and Molecular Properties,
2nd Ed., T.E.
Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews
are
available on this subject, such as by Wold, F., Posttranslational Covalent
Modification
ofProteins, B.C. Johnson, Ed., Academic Press, New York 1-12 (1983); Sei$er et
al.
(Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N. Y. Acad Sci.
663:48-62
( 1992)).
Accordingly, the transporter peptides of the present invention also encompass
derivatives or analogs in which a substituted amino acid residue is not one
encoded by
the genetic code, in which a substituent group is included, in which the
mature
transporter peptide is fused with another compound, such as a compound to
increase the
half life of the transporter peptide (for example, polyethylene glycol), or in
which the
additional amino acids are fused to the mature transporter peptide, such as a
leader or
secretory sequence or a sequence for purification of the mature transporter
peptide or a
pro-protein sequence.
Protein/Peptide Uses
The proteins of the present invention can be used in substantial and specific
assays related to the functional information provided in the Figures; to raise
antibodies or to elicit another immune response; as a reagent (including the
labeled
reagent) in assays designed to quantitatively determine levels of the protein
(or its
binding partner or ligand) in biological fluids; and as markers for tissues in
which the
corresponding protein is preferentially expressed (either constitutively or at
a
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particular stage of tissue differentiation or development or in a disease
state). Where
the protein binds or potentially binds to another protein or ligand (such as,
for
example, in a transporter-effector protein interaction or transporter-ligand
interaction),
the protein can be used to identify the binding partner/ligand so as to
develop a
5 system to identify inhibitors of the binding interaction. Any or all of
these uses are
capable of being developed into reagent grade or kit format for
commercialization as
commercial products.
Methods for performing the uses listed above are well known to those skilled
in the art. References disclosing such methods include "Molecular Cloning: A
10 Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook,
J., E.
F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to
Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
The potential uses of the peptides of the present invention are based
primarily
15 on the source of the protein as well as the class/action of the protein.
For example,
transporters isolated from humans and their human/mammalian orthologs serve as
targets for identifying agents for use in mammalian therapeutic applications,
e.g. a
human drug, particularly in modulating a biological or pathological response
in a cell
or tissue that expresses the transporter. Experimental data as provided in
Figure 1
20 indicates that the transporter proteins of the present invention are
expressed in humans
in the colon, kidney, and neuron tumors, as indicated by virtual northern blot
analysis.
PCR-based tissue screening panels also indicate expression in the kidney. A
large
percentage of pharmaceutical agents are being developed that modulate the
activity of
transporter proteins, particularly members of the sodium/chloride-dependent
organic
25 solute cotransporter subfamily (see Background of the Invention). The
structural and
functional information provided in the Background and Figures provide specific
and
substantial uses for the molecules of the present invention, particularly in
combination
with the expression information provided in Figure 1. Experimental data as
provided in
Figure 1 indicates expression in humans in the colon, kidney, and neuron
tumors. Such
uses can readily be determined using the information provided herein, that
known in
the art and routine experimentation.
The proteins of the present invention (including variants and fragments that
may
have been disclosed prior to the present invention) are useful for biological
assays
related to transporters that are related to members of the sodium/chloride-
dependent
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26
organic solute cotransporter subfamily. Such assays involve any of the known
transporter functions or activities or properties useful for diagnosis and
treatment of
transporter-related conditions that are specific for the subfamily of
transporters that the
one of the present invention belongs to, particularly in cells and tissues
that express the
transporter. Experimental data as provided in Figure 1 indicates that the
transporter
proteins of the present invention are expressed in humans in the colon,
kidney, and
neuron tumors, as indicated by virtual northern blot analysis. PCR-based
tissue
screening panels also indicate expression in the kidney. The proteins of the
present
invention are also useful in drug screening assays, in cell-based or cell-free
systems
((Hodgson, Biotechnology, 1992, Sept 10(9);973-80). Cell-based systems can be
native, i.e., cells that normally express the transporter, as a biopsy or
expanded in cell
culture. Experimental data as provided in Figure 1 indicates expression in
humans in the
colon, kidney, and neuron tumors. In an alternate embodiment, cell-based
assays
involve recombinant host cells expressing the transporter protein.
The polypeptides can be used to identify compounds that modulate transporter
activity of the protein in its natural state or an altered form that causes a
specific disease
or pathology associated with the transporter. Both the transporters of the
present
invention and appropriate variants and fragments can be used in high-
throughput screens
to assay candidate compounds for the ability to bind to the transporter. These
compounds can be further screened against a functional transporter to
determine the
effect of the compound on the transporter activity. Further, these compounds
can be
tested in animal or invertebrate systems to determine activity/effectiveness.
Compounds
can be identified that activate (agonist) or inactivate (antagonist) the
transporter to a
desired degree.
Further, the proteins of the present invention can be used to screen a
compound
for the ability to stimulate or inhibit interaction between the transporter
protein and a
molecule that normally interacts with the transporter protein, e.g. a
substrate or a
component of the signal pathway that the transporter protein normally
interacts (for
example, another transporter). Such assays typically include the steps of
combining the
transporter protein with a candidate compound under conditions that allow the
transporter protein, or fragment, to interact with the target molecule, and to
detect the
formation of a complex between the protein and the target or to detect the
biochemical
consequence of the interaction with the transporter protein and the target,
such as any of
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the associated effects of signal transduction such as changes in membrane
potential,
protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.
Candidate compounds include, for example, 1) peptides such as soluble
peptides,
including Ig-tailed fusion peptides and members of random peptide libraries
(see, e.g.,
Lam et al. , Nature 354:82-84 ( 1991 ); Houghten et al. , Nature 354:84-86 (
1991 )) and
combinatorial chemistry-derived molecular libraries made of D- and/or L-
configuration
amino acids; 2) phosphopeptides (e.g., members of random and partially
degenerate,
directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778
(1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric,
and single
chain antibodies as well as Fab, F(ab')2, Fab expression library fragments,
and epitope-
binding fragments of antibodies); and 4) small organic and inorganic molecules
(e.g.,
molecules obtained from combinatorial and natural product libraries).
One candidate compound is a soluble fragment of the receptor that competes for
ligand binding. Other candidate compounds include mutant transporters or
appropriate
fragments containing mutations that affect transporter function and thus
compete for
ligand. Accordingly, a fragment that competes for ligand, for example with a
higher
affinity, or a fragment that binds ligand but does not allow release, is
encompassed by
the invention.
The invention further includes other end point assays to identify compounds
that
modulate (stimulate or inhibit) transporter activity. The assays typically
involve an
assay of events in the signal transduction pathway that indicate transporter
activity.
Thus, the transport of a ligand, change in cell membrane potential, activation
of a
protein, a change in the expression of genes that are up- or down-regulated in
response to
the transporter protein dependent signal cascade can be assayed.
Any of the biological or biochemical functions mediated by the transporter can
be used as an endpoint assay. These include all of the biochemical or
biochemical/biological events described herein, in the references cited
herein,
incorporated by reference for these endpoint assay targets, and other
functions known to
those of ordinary skill in the art or that can be readily identified using the
information
provided in the Figures, particularly Figure 2. Specifically, a biological
function of a cell
or tissues that expresses the transporter can be assayed. Experimental data as
provided
in Figure 1 indicates that the transporter proteins of the present invention
are
expressed in humans in the colon, kidney, and neuron tumors, as indicated by
virtual
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northern blot analysis. PCR-based tissue screening panels also indicate
expression in
the kidney.
Binding and/or activating compounds can also be screened by using chimeric
transporter proteins in which the amino terminal extracellular domain, or
parts thereof,
the entire transmembrane domain or subregions, such as any of the seven
transmembrane segments or any of the intracellular or extracellular loops and
the
carboxy terminal intracellular domain, or parts thereof, can be replaced by
heterologous
domains or subregions. For example, a ligand-binding region can be used that
interacts
with a different ligand then that which is recognized by the native
transporter.
Accordingly, a different set of signal transduction components is available as
an end-
point assay for activation. This allows for assays to be performed in other
than the
specific host cell from which the transporter is derived.
The proteins of the present invention are also useful in competition binding
assays in methods designed to discover compounds that interact with the
transporter (e.g.
1 S binding partners and/or ligands). Thus, a compound is exposed to a
transporter
polypeptide under conditions that allow the compound to bind or to otherwise
interact
with the polypeptide. Soluble transporter polypeptide is also added to the
mixture. If the
test compound interacts with the soluble transporter polypeptide, it decreases
the amount
of complex formed or activity from the transporter target. This type of assay
is
particularly useful in cases in which compounds are sought that interact with
specific
regions of the transporter. Thus, the soluble polypeptide that competes with
the target
transporter region is designed to contain peptide sequences corresponding to
the region
of interest.
To perform cell free drug screening assays, it is sometimes desirable to
immobilize either the transporter protein, or fragment, or its target molecule
to facilitate
separation of complexes from uncomplexed forms of one or both of the proteins,
as well
as to accommodate automation of the assay.
Techniques for immobilizing proteins on matrices can be used in the drug
screening assays. In one embodiment, a fusion protein can be provided which
adds a
domain that allows the protein to be bound to a matrix. For example,
glutathione-S-
transferase fusion proteins can be adsorbed onto glutathione sepharose beads
(Sigma
Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which
are then
combined with the cell lysates (e.g., 35S-labeled) and the candidate compound,
and the
mixture incubated under conditions conducive to complex formation (e.g., at
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physiological conditions for salt and pH). Following incubation, the beads are
washed to
remove any unbound label, and the matrix immobilized and radiolabel determined
directly, or in the supernatant after the complexes are dissociated.
Alternatively, the
complexes can be dissociated from the matrix, separated by SDS-PAGE, and the
level of
S transporter-binding protein found in the bead fraction quantitated from the
gel using
standard electrophoretic techniques. For example, either the polypeptide or
its target
molecule can be immobilized utilizing conjugation of biotin and streptavidin
using
techniques well known in the art. Alternatively, antibodies reactive with the
protein but
which do not interfere with binding of the protein to its target molecule can
be
derivatized to the wells of the plate, and the protein trapped in the wells by
antibody
conjugation. Preparations of a transporter-binding protein and a candidate
compound are
incubated in the transporter protein-presenting wells and the amount of
complex trapped
in the well can be quantitated. Methods for detecting such complexes, in
addition to
those described above for the GST-immobilized complexes, include
immunodetection of
1 S complexes using antibodies reactive with the transporter protein target
molecule, or
which are reactive with transporter protein and compete with the target
molecule, as well
as enzyme-linked assays which rely on detecting an enzymatic activity
associated with
the target molecule.
Agents that modulate one of the transporters of the present invention can be
identified using one or more of the above assays, alone or in combination. It
is generally
preferable to use a cell-based or cell free system first and then confirm
activity in an
animal or other model system. Such model systems are well known in the art and
can
readily be employed in this context.
Modulators of transporter protein activity identified according to these drug
screening assays can be used to treat a subject with a disorder mediated by
the
transporter pathway, by treating cells or tissues that express the
transporter.
Experimental data as provided in Figure 1 indicates expression in humans in
the colon,
kidney, and neuron tumors. These methods of treatment include the steps of
administering a modulator of transporter activity in a pharmaceutical
composition to a
subject in need of such treatment, the modulator being identified as described
herein.
In yet another aspect of the invention, the transporter proteins can be used
as
"bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S.
Patent No.
5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol.
Chem.
268:12046-12054; Bartel et al. (1993) Biotechnigues 14:920-924; Iwabuchi et
al.
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(1993) Oncogene 8:1693-1696; and Brent W094/10300), to identify other
proteins,
which bind to or interact with the transporter and are involved in transporter
activity.
Such transporter-binding proteins are also likely to be involved in the
propagation of
signals by the transporter proteins or transporter targets as, for example,
downstream
5 elements of a transporter-mediated signaling pathway. Alternatively, such
transporter-binding proteins are likely to be transporter inhibitors.
The two-hybrid system is based on the modular nature of most transcription
factors, which consist of separable DNA-binding and activation domains.
Briefly, the
assay utilizes two different DNA constructs. In one construct, the gene that
codes for
10 a transporter protein is fused to a gene encoding the DNA binding domain of
a known
transcription factor (e.g., GAL-4). In the other construct, a DNA sequence,
from a
library of DNA sequences, that encodes an unidentified protein ("prey" or
"sample")
is fused to a gene that codes for the activation domain of the known
transcription
factor. If the "bait" and the "prey" proteins are able to interact, in vivo,
forming a
15 transporter-dependent complex, the DNA-binding and activation domains of
the
transcription factor are brought into close proximity. This proximity allows
transcription of a reporter gene (e.g., LacZ) which is operably linked to a
transcriptional regulatory site responsive to the transcription factor.
Expression of the
reporter gene can be detected and cell colonies containing the functional
transcription
20 factor can be isolated and used to obtain the cloned gene which encodes the
protein
which interacts with the transporter protein.
This invention further pertains to novel agents identified by the above-
described screening assays. Accordingly, it is within the scope of this
invention to
further use an agent identified as described herein in an appropriate animal
model.
25 For example, an agent identified as described herein (e.g., a transporter-
modulating
agent, an antisense transporter nucleic acid molecule, a transporter-specific
antibody,
or a transporter-binding partner) can be used in an animal or other model to
determine
the efficacy, toxicity, or side effects of treatment with such an agent.
Alternatively,
an agent identified as described herein can be used in an animal or other
model to
30 determine the mechanism of action of such an agent. Furthermore, this
invention
pertains to uses of novel agents identified by the above-described screening
assays for
treatments as described herein.
The transporter proteins of the present invention are also useful to provide a
target for diagnosing a disease or predisposition to disease mediated by the
peptide.
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Accordingly, the invention provides methods for detecting the presence, or
levels of, the
protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data
as
provided in Figure 1 indicates expression in humans in the colon, kidney, and
neuron
tumors. The method involves contacting a biological sample with a compound
capable
of interacting with the transporter protein such that the interaction can be
detected. Such
an assay can be provided in a single detection format or a mufti-detection
format such as
an antibody chip array.
One agent for detecting a protein in a sample is an antibody capable of
selectively binding to protein. A biological sample includes tissues, cells
and biological
fluids isolated from a subject, as well as tissues, cells and fluids present
within a subject.
The peptides of the present invention also provide targets for diagnosing
active
protein activity, disease, or predisposition to disease, in a patient having a
variant
peptide, particularly activities and conditions that are known for other
members of the
family of proteins to which the present one belongs. Thus, the peptide can be
isolated
from a biological sample and assayed for the presence of a genetic mutation
that results
in aberrant peptide. This includes amino acid substitution, deletion,
insertion,
rearrangement, (as the result of aberrant splicing events), and inappropriate
post-
translational modification. Analytic methods include altered electrophoretic
mobility,
altered tryptic peptide digest, altered transporter activity in cell-based or
cell-free assay,
alteration in ligand or antibody-binding pattern, altered isoelectric point,
direct amino
acid sequencing, and any other of the known assay techniques useful for
detecting
mutations in a protein. Such an assay can be provided in a single detection
format or a
mufti-detection format such as an antibody chip array.
In vitro techniques for detection of peptide include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence using a detection reagent, such as an antibody or protein
binding
agent. Alternatively, the peptide can be detected in vivo in a subject by
introducing into
the subject a labeled anti-peptide antibody or other types of detection agent.
For
example, the antibody can be labeled with a radioactive marker whose presence
and
location in a subject can be detected by standard imaging techniques.
Particularly useful
are methods that detect the allelic variant of a peptide expressed in a
subject and methods
which detect fragments of a peptide in a sample.
The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics
deal with clinically significant hereditary variations in the response to
drugs due to
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altered drug disposition and abnormal action in affected persons. See, e.g.,
Eichelbaum,
M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linden M.W.
(Clin.
Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result
in severe
toxicity of therapeutic drugs in certain individuals or therapeutic failure of
drugs in
certain individuals as a result of individual variation in metabolism. Thus,
the genotype
of the individual can determine the way a therapeutic compound acts on the
body or the
way the body metabolizes the compound. Further, the activity of drug
metabolizing
enzymes effects both the intensity and duration of drug action. Thus, the
pharmacogenomics of the individual permit the selection of effective compounds
and
effective dosages of such compounds for prophylactic or therapeutic treatment
based on
the individual's genotype. The discovery of genetic polymorphisms in some drug
metabolizing enzymes has explained why some patients do not obtain the
expected drug
effects, show an exaggerated drug effect, or experience serious toxicity from
standard
drug dosages. Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic
polymorphism may lead to allelic protein variants of the transporter protein
in which one
or more of the transporter functions in one population is different from those
in another
population. The peptides thus allow a target to ascertain a genetic
predisposition that can
affect treatment modality. Thus, in a ligand-based treatment, polymorphism may
give
rise to amino terminal extracellular domains and/or other ligand-binding
regions that are
more or less active in ligand binding, and transporter activation.
Accordingly, ligand
dosage would necessarily be modified to maximize the therapeutic effect within
a given
population containing a polymorphism. As an alternative to genotyping,
specific
polymorphic peptides could be identified.
The peptides are also useful for treating a disorder characterized by an
absence
of, inappropriate, or unwanted expression of the protein. Experimental data as
provided
in Figure 1 indicates expression in humans in the colon, kidney, and neuron
tumors.
Accordingly, methods for treatment include the use of the transporter protein
or
fragments.
Antibodies
The invention also provides antibodies that selectively bind to one of the
peptides of the present invention, a protein comprising such a peptide, as
well as variants
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and fragments thereof. As used herein, an antibody selectively binds a target
peptide
when it binds the target peptide and does not significantly bind to unrelated
proteins. An
antibody is still considered to selectively bind a peptide even if it also
binds to other
proteins that are not substantially homologous with the target peptide so long
as such
proteins share homology with a fragment or domain of the peptide target of the
antibody.
In this case, it would be understood that antibody binding to the peptide is
still selective
despite some degree of cross-reactivity.
As used herein, an antibody is defined in terms consistent with that
recognized
within the art: they are mufti-subunit proteins produced by a mammalian
organism in
response to an antigen challenge. The antibodies of the present invention
include
polyclonal antibodies and monoclonal antibodies, as well as fragments of such
antibodies, including, but not limited to, Fab or F(ab')z, and Fv fragments.
Many methods are known for generating and/or identifying antibodies to a given
target peptide. Several such methods are described by Harlow, Antibodies, Cold
Spring
Harbor Press, (1989).
In general, to generate antibodies, an isolated peptide is used as an
immunogen
and is administered to a mammalian organism, such as a rat, rabbit or mouse.
The full-
length protein, an antigenic peptide fragment or a fusion protein can be used.
Particularly important fragments are those covering functional domains, such
as the
domains identified in Figure 2, and domain of sequence homology or divergence
amongst the family, such as those that can readily be identified using protein
alignment
methods and as presented in the Figures.
Antibodies are preferably prepared from regions or discrete fragments of the
transporter proteins. Antibodies can be prepared from any region of the
peptide as
described herein. However, preferred regions will include those involved in
function/activity and/or transporter/binding partner interaction. Figure 2 can
be used
to identify particularly important regions while sequence alignment can be
used to
identify conserved and unique sequence fragments.
An antigenic fragment will typically comprise at least 8 contiguous amino acid
residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16
or more
amino acid residues. Such fragments can be selected on a physical property,
such as
fragments correspond to regions that are located on the surface of the
protein, e.g.,
hydrophilic regions or can be selected based on sequence uniqueness (see
Figure 2).
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Detection on an antibody of the present invention can be facilitated by
coupling
(i.e., physically linking) the antibody to a detectable substance. Examples of
detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive materials.
Examples of
suitable enzymes include horseradish peroxidase, alkaline phosphatase, (3-
galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group complexes
include
streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes luminol; examples of bioluminescent materials
include
luciferase, luciferin, and aequorin, and examples of suitable radioactive
material include
~2sh i3ih ass or 3H.
Antibody Uses
The antibodies can be used to isolate one of the proteins of the present
invention
by standard techniques, such as amity chromatography or immunoprecipitation.
The
antibodies can facilitate the purification of the natural protein from cells
and
recombinantly produced protein expressed in host cells. In addition, such
antibodies are
useful to detect the presence of one of the proteins of the present invention
in cells or
tissues to determine the pattern of expression of the protein among various
tissues in an
organism and over the course of normal development. Experimental data as
provided
in Figure 1 indicates that the transporter proteins of the present invention
are
expressed in humans in the colon, kidney, and neuron tumors, as indicated by
virtual
northern blot analysis. PCR-based tissue screening panels also indicate
expression in
the kidney. Further, such antibodies can be used to detect protein in situ, in
vitro, or in a
cell lysate or supernatant in order to evaluate the abundance and pattern of
expression.
Also, such antibodies can be used to assess abnormal tissue distribution or
abnormal
expression during development or progression of a biological condition.
Antibody
detection of circulating fragments of the full length protein can be used to
identify
turnover.
Further, the antibodies can be used to assess expression in disease states
such as
in active stages of the disease or in an individual with a predisposition
toward disease
related to the protein's function. When a disorder is caused by an
inappropriate tissue
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distribution, developmental expression, level of expression of the protein, or
expressed/processed form, the antibody can be prepared against the normal
protein.
Experimental data as provided in Figure 1 indicates expression in humans in
the colon,
kidney, and neuron tumors. If a disorder is characterized by a specific
mutation in the
5 protein, antibodies specific for this mutant protein can be used to assay
for the presence
of the specific mutant protein.
The antibodies can also be used to assess normal and aberrant subcellular
localization of cells in the various tissues in an organism. Experimental data
as provided
in Figure 1 indicates expression in humans in the colon, kidney, and neuron
tumors. The
10 diagnostic uses can be applied, not only in genetic testing, but also in
monitoring a
treatment modality. Accordingly, where treatment is ultimately aimed at
correcting
expression level or the presence of aberrant sequence and aberrant tissue
distribution or
developmental expression, antibodies directed against the protein or relevant
fragments
can be used to monitor therapeutic efficacy.
15 Additionally, antibodies are useful in pharmacogenomic analysis. Thus,
antibodies prepared against polymorphic proteins can be used to identify
individuals that
require modified treatment modalities. The antibodies are also useful as
diagnostic tools
as an immunological marker for aberrant protein analyzed by electrophoretic
mobility,
isoelectric point, tryptic peptide digest, and other physical assays known to
those in the
20 art.
The antibodies are also useful for tissue typing. Experimental data as
provided
in Figure 1 indicates expression in humans in the colon, kidney, and neuron
tumors.
Thus, where a specific protein has been correlated with expression in a
specific tissue,
antibodies that are specific for this protein can be used to identify a tissue
type.
25 The antibodies are also useful for inhibiting protein function, for
example,
blocking the binding of the transporter peptide to a binding partner such as a
ligand or
protein binding partner. These uses can also be applied in a therapeutic
context in which
treatment involves inhibiting the protein's function. An antibody can be used,
for
example, to block binding, thus modulating (agonizing or antagonizing) the
peptides
30 activity. Antibodies can be prepared against specific fragments containing
sites required
for function or against intact protein that is associated with a cell or cell
membrane. See
Figure 2 for structural information relating to the proteins of the present
invention.
The invention also encompasses kits for using antibodies to detect the
presence
of a protein in a biological sample. The kit can comprise antibodies such as a
labeled or
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36
labelable antibody and a compound or agent for detecting protein in a
biological sample;
means for determining the amount of protein in the sample; means for comparing
the
amount of protein in the sample with a standard; and instructions for use.
Such a kit can
be supplied to detect a single protein or epitope or can be configured to
detect one of a
multitude of epitopes, such as in an antibody detection array. Arrays are
described in
detail below for nucleic acid arrays and similar methods have been developed
for
antibody arrays.
Nucleic Acid Molecules
The present invention further provides isolated nucleic acid molecules that
encode a transporter peptide or protein of the present invention (cDNA,
transcript and
genomic sequence). Such nucleic acid molecules will consist of, consist
essentially of,
or comprise a nucleotide sequence that encodes one of the transporter peptides
of the
present invention, an allelic variant thereof, or an ortholog or paralog
thereof.
As used herein, an "isolated" nucleic acid molecule is one that is separated
from
other nucleic acid present in the natural source of the nucleic acid.
Preferably, an
"isolated" nucleic acid is free of sequences that naturally flank the nucleic
acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the genomic
DNA of the
organism from which the nucleic acid is derived. However, there can be some
flanking
nucleotide sequences, for example up to about SKB, 4KB, 3KB, 2KB, or 1KB or
less,
particularly contiguous peptide encoding sequences and peptide encoding
sequences
within the same gene but separated by introns in the genomic sequence. The
important
point is that the nucleic acid is isolated from remote and unimportant
flanking sequences
such that it can be subjected to the specific manipulations described herein
such as
recombinant expression, preparation of probes and primers, and other uses
specific to the
nucleic acid sequences.
Moreover, an "isolated" nucleic acid molecule, such as a transcript/cDNA
molecule, can be substantially free of other cellular material, or culture
medium when
produced by recombinant techniques, or chemical precursors or other chemicals
when
chemically synthesized. However, the nucleic acid molecule can be fused to
other
coding or regulatory sequences and still be considered isolated.
For example, recombinant DNA molecules contained in a vector are considered
isolated. Further examples of isolated DNA molecules include recombinant DNA
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molecules maintained in heterologous host cells or purified (partially or
substantially)
DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro
RNA
transcripts of the isolated DNA molecules of the present invention. Isolated
nucleic acid
molecules according to the present invention further include such molecules
produced
synthetically.
Accordingly, the present invention provides nucleic acid molecules that
consist
of the nucleotide sequence shown in Figure 1 or 3 (SEQ ID NO:1, transcript
sequence
and SEQ ID N0:3, genomic sequence), or any nucleic acid molecule that encodes
the
protein provided in Figure 2, SEQ ID N0:2. A nucleic acid molecule consists of
a
nucleotide sequence when the nucleotide sequence is the complete nucleotide
sequence
of the nucleic acid molecule.
The present invention further provides nucleic acid molecules that consist
essentially of the nucleotide sequence shown in Figure 1 or 3 (SEQ ID NO:1,
transcript
sequence and SEQ ID N0:3, genomic sequence), or any nucleic acid molecule that
encodes the protein provided in Figure 2, SEQ ID N0:2. A nucleic acid molecule
consists essentially of a nucleotide sequence when such a nucleotide sequence
is present
with only a few additional nucleic acid residues in the final nucleic acid
molecule.
The present invention further provides nucleic acid molecules that comprise
the
nucleotide sequences shown in Figure 1 or 3 (SEQ ID NO:1, transcript sequence
and
SEQ ID N0:3, genomic sequence), or any nucleic acid molecule that encodes the
protein
provided in Figure 2, SEQ >D N0:2. A nucleic acid molecule comprises a
nucleotide
sequence when the nucleotide sequence is at least part of the final nucleotide
sequence of
the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be
only the
nucleotide sequence or have additional nucleic acid residues, such as nucleic
acid
residues that are naturally associated with it or heterologous nucleotide
sequences. Such
a nucleic acid molecule can have a few additional nucleotides or can comprise
several
hundred or more additional nucleotides. A brief description of how various
types of
these nucleic acid molecules can be readily made/isolated is provided below.
In Figures l and 3, both coding and non-coding sequences are provided.
Because of the source of the present invention, humans genomic sequence
(Figure 3)
and cDNA/transcript sequences (Figure 1), the nucleic acid molecules in the
Figures
will contain genomic intronic sequences, 5' and 3' non-coding sequences, gene
regulatory regions and non-coding intergenic sequences. In general such
sequence
features are either noted in Figures l and 3 or can readily be identified
using
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computational tools known in the art. As discussed below, some of the non-
coding
regions, particularly gene regulatory elements such as promoters, are useful
for a
variety of purposes, e.g. control of heterologous gene expression, target for
identifying gene activity modulating compounds, and axe particularly claimed
as
fragments of the genomic sequence provided herein.
The isolated nucleic acid molecules can encode the mature protein plus
additional amino or carboxyl-terminal amino acids, or amino acids interior to
the mature
peptide (when the mature form has more than one peptide chain, for instance).
Such
sequences may play a role in processing of a protein from precursor to a
mature form,
facilitate protein trafficking, prolong or shorten protein half life or
facilitate
manipulation of a protein for assay or production, among other things. As
generally is
the case in situ, the additional amino acids may be processed away from the
mature
protein by cellular enzymes.
As mentioned above, the isolated nucleic acid molecules include, but are not
I 5 limited to, the sequence encoding the transporter peptide alone, the
sequence encoding
the mature peptide and additional coding sequences, such as a leader or
secretory
sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the
mature
peptide, with or without the additional coding sequences, plus additional non-
coding
sequences, for example introns and non-coding 5' and 3' sequences such as
transcribed
but non-translated sequences that play a role in transcription, mRNA
processing
(including splicing and polyadenylation signals), ribosome binding and
stability of
mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence
encoding, for example, a peptide that facilitates purification.
Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in
the form DNA, including cDNA and genomic DNA obtained by cloning or produced
by
chemical synthetic techniques or by a combination thereof. The nucleic acid,
especially
DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid
can be
the coding strand (sense strand) or the non-coding strand (anti-sense strand).
The invention further provides nucleic acid molecules that encode fragments of
the peptides of the present invention as well as nucleic acid molecules that
encode
obvious variants of the transporter proteins of the present invention that are
described
above. Such nucleic acid molecules may be naturally occurnng, such as allelic
variants
(same locus), paralogs (different locus), and orthologs (different organism),
or may be
constructed by recombinant DNA methods or by chemical synthesis. Such non-
naturally
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occurring variants may be made by mutagenesis techniques, including those
applied to
nucleic acid molecules, cells, or organisms. Accordingly, as discussed above,
the
variants can contain nucleotide substitutions, deletions, inversions and
insertions.
Variation can occur in either or both the coding and non-coding regions. The
variations
can produce both conservative and non-conservative amino acid substitutions.
The present invention further provides non-coding fragments of the nucleic
acid
molecules provided in Figures 1 and 3. Preferred non-coding fragments include,
but are
not limited to, promoter sequences, enhancer sequences, gene modulating
sequences and
gene termination sequences. Such fragments are useful in controlling
heterologous gene
expression and in developing screens to identify gene-modulating agents. A
promoter
can readily be identified as being 5' to the ATG start site in the genomic
sequence
provided in Figure 3.
A fragment comprises a contiguous nucleotide sequence greater than 12 or more
nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500
nucleotides in
length. The length of the fragment will be based on its intended use. For
example, the
fragment can encode epitope bearing regions of the peptide, or can be useful
as DNA
probes and primers. Such fragments can be isolated using the known nucleotide
sequence to synthesize an oligonucleotide probe. A labeled probe can then be
used to
screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in PCR
reactions to
clone specific regions of gene.
A probe/primer typically comprises substantially a purified oligonucleotide or
oligonucleotide pair. The oligonucleotide typically comprises a region of
nucleotide
sequence that hybridizes under stringent conditions to at least about 12, 20,
25, 40, SO or
more consecutive nucleotides.
Orthologs, homologs, and allelic variants can be identified using methods well
known in the art. As described in the Peptide Section, these variants comprise
a
nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-
90%, and
more typically at least about 90-95% or more homologous to the nucleotide
sequence
shown in the Figure sheets or a fragment of this sequence. Such nucleic acid
molecules
can readily be identified as being able to hybridize under moderate to
stringent
conditions, to the nucleotide sequence shown in the Figure sheets or a
fragment of the
sequence. Allelic variants can readily be determined by genetic locus of the
encoding
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gene. The gene encoding the novel transporter protein of the present invention
is located
on human chromosome 5.
Figure 3 provides information on SNPs that have been found in the gene
encoding the transporter protein of the present invention. SNPs were
identified at 23
5 different nucleotide positions. Changes in the amino acid sequence caused by
these
SNPs can readily be determined using the universal genetic code and the
protein
sequence provided in Figure 2 as a reference. These SNPs may also affect
control/regulatory elements.
As used herein, the term "hybridizes under stringent conditions" is intended
to
10 describe conditions for hybridization and washing under which nucleotide
sequences
encoding a peptide at least 60-70% homologous to each other typically remain
hybridized to each other. The conditions can be such that sequences at least
about 60%,
at least about 70%, or at least about 80% or more homologous to each other
typically
remain hybridized to each other. Such stringent conditions are known to those
skilled in
15 the art and can be found in Current Protocols in Molecular Biology, John
Wiley & Sons,
N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions
are
hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45C,
followed by one
or more washes in 0.2 X SSC, 0.1 % SDS at 50-65C. Examples of moderate to low
stringency hybridization conditions are well known in the art.
Nucleic Acid Molecule Uses
The nucleic acid molecules of the present invention are useful for probes,
primers, chemical intermediates, and in biological assays. The nucleic acid
molecules
are useful as a hybridization probe for messenger RNA, transcripbcDNA and
genomic
DNA to isolate full-length cDNA and genomic clones encoding the peptide
described in
Figure 2 and to isolate cDNA and genomic clones that correspond to variants
(alleles,
orthologs, etc.) producing the same or related peptides shown in Figure 2. As
illustrated
in Figure 3, SNPs were identified at 23 different nucleotide positions.
The probe can correspond to any sequence along the entire length of the
nucleic
acid molecules provided in the Figures. Accordingly, it could be derived from
5'
noncoding regions, the coding region, and 3' noncoding regions. However, as
discussed,
fragments are not to be construed as encompassing fragments disclosed prior to
the
present invention.
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The nucleic acid molecules are also useful as primers for PCR to amplify any
given region of a nucleic acid molecule and are useful to synthesize antisense
molecules
of desired length and sequence.
The nucleic acid molecules are also useful for constructing recombinant
vectors.
S Such vectors include expression vectors that express a portion of, or all
of, the peptide
sequences. Vectors also include insertion vectors, used to integrate into
another nucleic
acid molecule sequence, such as into the cellular genome, to alter in situ
expression of a
gene and/or gene product. For example, an endogenous coding sequence can be
replaced via homologous recombination with all or part of the coding region
containing
one or more specifically introduced mutations.
The nucleic acid molecules are also useful for expressing antigenic portions
of
the proteins.
The nucleic acid molecules are also useful as probes for determining the
chromosomal positions of the nucleic acid molecules by means of in situ
hybridization
methods. The gene encoding the novel transporter protein of the present
invention is
located on human chromosome 5.
The nucleic acid molecules are also useful in making vectors containing the
gene
regulatory regions of the nucleic acid molecules of the present invention.
The nucleic acid molecules are also useful for designing ribozymes
corresponding to all, or a part, of the mRNA produced from the nucleic acid
molecules
described herein.
The nucleic acid molecules are also useful for making vectors that express
part,
or all, of the peptides.
The nucleic acid molecules are also useful for constructing host cells
expressing
a part, or all, of the nucleic acid molecules and peptides.
The nucleic acid molecules are also useful for constructing transgenic animals
expressing all, or a part, of the nucleic acid molecules and peptides.
The nucleic acid molecules are also useful as hybridization probes for
determining the presence, level, form and distribution of nucleic acid
expression.
Experimental data as provided in Figure 1 indicates that the transporter
proteins of the
present invention are expressed in humans in the colon, kidney, and neuron
tumors, as
indicated by virtual northern blot analysis. PCR-based tissue screening panels
also
indicate expression in the kidney.
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Accordingly, the probes can be used to detect the presence of, or to determine
levels of, a specific nucleic acid molecule in cells, tissues, and in
organisms. The nucleic
acid whose level is determined can be DNA or RNA. Accordingly, probes
corresponding to the peptides described herein can be used to assess
expression and/or
S gene copy number in a given cell, tissue, or organism. These uses are
relevant for
diagnosis of disorders involving an increase or decrease in transporter
protein expression
relative to normal results.
In vitro techniques for detection of mRNA include Northern hybridizations and
in situ hybridizations. In vitro techniques for detecting DNA include Southern
hybridizations and in situ hybridization.
Probes can be used as a part of a diagnostic test kit for identifying cells or
tissues
that express a transporter protein, such as by measuring a level of a
transporter-encoding
nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or
determining if a transporter gene has been mutated. Experimental data as
provided in
1 S Figure 1 indicates that the transporter proteins of the present invention
are expressed
in humans in the colon, kidney, and neuron tumors, as indicated by virtual
northern
blot analysis. PCR-based tissue screening panels also indicate expression in
the
kidney.
Nucleic acid expression assays are useful for drug screening to identify
compounds that modulate transporter nucleic acid expression.
The invention thus provides a method for identifying a compound that can be
used to treat a disorder associated with nucleic acid expression of the
transporter gene,
particularly biological and pathological processes that are mediated by the
transporter in
cells and tissues that express it. Experimental data as provided in Figure 1
indicates
expression in humans in the colon, kidney, and neuron tumors. The method
typically
includes assaying the ability of the compound to modulate the expression of
the
transporter nucleic acid and thus identifying a compound that can be used to
treat a
disorder characterized by undesired transporter nucleic acid expression. The
assays can
be performed in cell-based and cell-free systems. Cell-based assays include
cells
naturally expressing the transporter nucleic acid or recombinant cells
genetically
engineered to express specific nucleic acid sequences.
The assay for transporter nucleic acid expression can involve direct assay of
nucleic acid levels, such as mRNA levels, or on collateral compounds involved
in the
signal pathway. Further, the expression of genes that are up- or down-
regulated in
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response to the transporter protein signal pathway can also be assayed. In
this
embodiment the regulatory regions of these genes can be operably linked to a
reporter
gene such as luciferase.
Thus, modulators of transporter gene expression can be identified in a method
wherein a cell is contacted with a candidate compound and the expression of
mRNA
determined. The level of expression of transporter mRNA in the presence of the
candidate compound is compared to the level of expression of transporter mRNA
in the
absence of the candidate compound. The candidate compound can then be
identified as
a modulator of nucleic acid expression based on this comparison and be used,
for
example to treat a disorder characterized by aberrant nucleic acid expression.
When
expression of mRNA is statistically significantly greater in the presence of
the candidate
compound than in its absence, the candidate compound is identified as a
stimulator of
nucleic acid expression. When nucleic acid expression is statistically
significantly less
in the presence of the candidate compound than in its absence, the candidate
compound
is identified as an inhibitor of nucleic acid expression.
The invention further provides methods of treatment, with the nucleic acid as
a
target, using a compound identified through drug screening as a gene modulator
to
modulate transporter nucleic acid expression in cells and tissues that express
the
transporter. Experimental data as provided in Figure 1 indicates that the
transporter
proteins of the present invention are expressed in humans in the colon,
kidney, and
neuron tumors, as indicated by virtual northern blot analysis. PCR-based
tissue
screening panels also indicate expression in the kidney. Modulation includes
both up-
regulation (i.e. activation or agonization) or down-regulation (suppression or
antagonization) or nucleic acid expression.
Alternatively, a modulator for transporter nucleic acid expression can be a
small
molecule or drug identified using the screening assays described herein as
long as the
drug or small molecule inhibits the transporter nucleic acid expression in the
cells and
tissues that express the protein. Experimental data as provided in Figure 1
indicates
expression in humans in the colon, kidney, and neuron tumors.
The nucleic acid molecules are also useful for monitoring the effectiveness of
modulating compounds on the expression or activity of the transporter gene in
clinical
trials or in a treatment regimen. Thus, the gene expression pattern can serve
as a
barometer for the continuing effectiveness of treatment with the compound,
particularly
with compounds to which a patient can develop resistance. The gene expression
pattern
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can also serve as a marker indicative of a physiological response of the
affected cells to
the compound. Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative compounds
to which
the patient has not become resistant. Similarly, if the level of nucleic acid
expression
falls below a desirable level, administration of the compound could be
commensurately
decreased.
The nucleic acid molecules are also useful in diagnostic assays for
qualitative
changes in transporter nucleic acid expression, and particularly in
qualitative changes
that lead to pathology. The nucleic acid molecules can be used to detect
mutations in
transporter genes and gene expression products such as mRNA. The nucleic acid
molecules can be used as hybridization probes to detect naturally occurring
genetic
mutations in the transporter gene and thereby to determine whether a subject
with the
mutation is at risk for a disorder caused by the mutation. Mutations include
deletion,
addition, or substitution of one or more nucleotides in the gene, chromosomal
rearrangement, such as inversion or transposition, modification of genomic
DNA, such
as aberrant methylation patterns or changes in gene copy number, such as
amplification.
Detection of a mutated form of the transporter gene associated with a
dysfunction
provides a diagnostic tool for an active disease or susceptibility to disease
when the
disease results from overexpression, underexpression, or altered expression of
a
transporter protein.
Individuals carrying mutations in the transporter gene can be detected at the
nucleic acid level by a variety of techniques. Figure 3 provides information
on SNPs
that have been found in the gene encoding the transporter protein of the
present
invention. SNPs were identified at 23 different nucleotide positions. Changes
in the
amino acid sequence caused by these SNPs can readily be determined using the
universal genetic code and the protein sequence provided in Figure 2 as a
reference.
These SNPs may also affect control/regulatory elements. The gene encoding the
novel
transporter protein of the present invention is located on human chromosome 5.
Genomic DNA can be analyzed directly or can be amplified by using PCR prior to
analysis. RNA or cDNA can be used in the same way. In some uses, detection of
the
mutation involves the use of a probe/primer in a polymerase chain reaction
(PCR) (see,
e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE
PCR, or,
alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et
al., Science
241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter
of
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which can be particularly useful for detecting point mutations in the gene
(see Abravaya
et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the
steps of
collecting a sample of cells from a patient, isolating nucleic acid (e.g.,
genomic, mRNA
or both) from the cells of the sample, contacting the nucleic acid sample with
one or
5 more primers which specifically hybridize to a gene under conditions such
that
hybridization and amplification of the gene (if present) occurs, and detecting
the
presence or absence of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample. Deletions
and
insertions can be detected by a change in size of the amplified product
compared to the
10 normal genotype. Point mutations can be identified by hybridizing amplified
DNA to
normal RNA or antisense DNA sequences.
Alternatively, mutations in a transporter gene can be directly identified, for
example, by alterations in restriction enzyme digestion patterns determined by
gel
electrophoresis.
15 Further, sequence-specific ribozymes (LJ.S. Patent No. 5,498,531) can be
used to
score for the presence of specific mutations by development or loss of a
ribozyme
cleavage site. Perfectly matched sequences can be distinguished from
mismatched
sequences by nuclease cleavage digestion assays or by differences in melting
temperature.
20 Sequence changes at specific locations can also be assessed by nuclease
protection assays such as RNase and S 1 protection or the chemical cleavage
method.
Furthermore, sequence differences between a mutant transporter gene and a wild-
type
gene can be determined by direct DNA sequencing. A variety of automated
sequencing
procedures can be utilized when performing the diagnostic assays (Naeve, C.W.,
(1995)
25 Biotechniques 19:448), including sequencing by mass spectrometry (see,
e.g., PCT
International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr.
36:127-162
(1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).
Other methods for detecting mutations in the gene include methods in which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or
30 RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al.,
PNAS
85:4397 (1988); Saleeba et al., Meth. Enrymol. 217:286-295 (1992)),
electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS
86:2766
(1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,
Genet. Anal.
Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in
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46
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing
gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples
of other
techniques for detecting point mutations include selective oligonucleotide
hybridization,
selective amplification, and selective primer extension.
The nucleic acid molecules are also useful for testing an individual for a
genotype that while not necessarily causing the disease, nevertheless affects
the
treatment modality. Thus, the nucleic acid molecules can be used to study the
relationship between an individual's genotype and the individual's response to
a
compound used for treatment (pharmacogenomic relationship). Accordingly, the
nucleic
acid molecules described herein can be used to assess the mutation content of
the
transporter gene in an individual in order to select an appropriate compound
or dosage
regimen for treatment. Figure 3 provides information on SNPs that have been
found in
the gene encoding the transporter protein of the present invention. SNPs were
identified at 23 different nucleotide positions. Changes in the amino acid
sequence
caused by these SNPs can readily be determined using the universal genetic
code and
the protein sequence provided in Figure 2 as a reference. These SNPs may also
affect
control/regulatory elements.
Thus nucleic acid molecules displaying genetic variations that affect
treatment
provide a diagnostic target that can be used to tailor treatment in an
individual.
Accordingly, the production of recombinant cells and animals containing these
polymorphisms allow effective clinical design of treatment compounds and
dosage
regimens.
The nucleic acid molecules are thus useful as antisense constructs to control
transporter gene expression in cells, tissues, and organisms. A DNA antisense
nucleic
acid molecule is designed to be complementary to a region of the gene involved
in
transcription, preventing transcription and hence production of transporter
protein. An
antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and
thus
block translation of mRNA into transporter protein.
Alternatively, a class of antisense molecules can be used to inactivate mRNA
in
order to decrease expression of transporter nucleic acid. Accordingly, these
molecules
can treat a disorder characterized by abnormal or undesired transporter
nucleic acid
expression. This technique involves cleavage by means of ribozymes containing
nucleotide sequences complementary to one or more regions in the mRNA that
attenuate
the ability of the mRNA to be translated. Possible regions include coding
regions and
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particularly coding regions corresponding to the catalytic and other
functional activities
of the transporter protein, such as ligand binding.
The nucleic acid molecules also provide vectors for gene therapy in patients
containing cells that are aberrant in transporter gene expression. Thus,
recombinant
cells, which include the patient's cells that have been engineered ex vivo and
returned to
the patient, are introduced into an individual where the cells produce the
desired
transporter protein to treat the individual.
The invention also encompasses kits for detecting the presence of a
transporter
nucleic acid in a biological sample. Experimental data as provided in Figure 1
indicates that the transporter proteins of the present invention are expressed
in humans
in the colon, kidney, and neuron tumors, as indicated by virtual northern blot
analysis.
PCR-based tissue screening panels also indicate expression in the kidney. For
example, the kit can comprise reagents such as a labeled or labelable nucleic
acid or
agent capable of detecting transporter nucleic acid in a biological sample;
means for
determining the amount of transporter nucleic acid in the sample; and means
for
comparing the amount of transporter nucleic acid in the sample with a
standard. The
compound or agent can be packaged in a suitable container. The kit can further
comprise instructions for using the kit to detect transporter protein mRNA or
DNA.
Nucleic Acid Arrays
The present invention further provides nucleic acid detection kits, such as
arrays or microarrays of nucleic acid molecules that are based on the sequence
information provided in Figures l and 3 (SEQ ID NOS:1 and 3).
As used herein "Arrays" or "Microarrays" refers to an array of distinct
polynucleotides or oligonucleotides synthesized on a substrate, such as paper,
nylon
or other type of membrane, filter, chip, glass slide, or any other suitable
solid support.
In one embodiment, the microarray is prepared and used according to the
methods
described in US Patent 5,837,832, Chee et al., PCT application W095/11995
(Chee et
al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,
M. et al.
(1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated
herein
in their entirety by reference. In other embodiments, such arrays are produced
by the
methods described by Brown et al., US Patent No. 5,807,522.
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The microarray or detection kit is preferably composed of a large number of
unique, single-stranded nucleic acid sequences, usually either synthetic
antisense
oligonucleotides or fragments of cDNAs, fixed to a solid support. The
oligonucleotides are preferably about 6-60 nucleotides in length, more
preferably 1 S-
30 nucleotides in length, and most preferably about 20-25 nucleotides in
length. For a
certain type of microarray or detection kit, it may be preferable to use
oligonucleotides that are only 7-20 nucleotides in length. The microarray or
detection
kit may contain oligonucleotides that cover the known S', or 3', sequence,
sequential
oligonucleotides that cover the full length sequence; or unique
oligonucleotides
selected from particular areas along the length of the sequence.
Polynucleotides used
in the microarray or detection kit may be oligonucleotides that are specific
to a gene
or genes of interest.
In order to produce oligonucleotides to a known sequence for a microarray or
detection kit, the genes) of interest (or an ORF identified from the contigs
of the
present invention) is typically examined using a computer algorithm which
starts at
the 5' or at the 3' end of the nucleotide sequence. Typical algorithms will
then
identify oligomers of defined length that are unique to the gene, have a GC
content
within a range suitable for hybridization, and lack predicted secondary
structure that
may interfere with hybridization. In certain situations it may be appropriate
to use
pairs of oligonucleotides on a microarray or detection kit. The "pairs" will
be
identical, except for one nucleotide that preferably is located in the center
of the
sequence. The second oligonucleotide in the pair (mismatched by one) serves as
a
control. The number of oligonucleotide pairs may range from two to one
million.
The oligomers are synthesized at designated areas on a substrate using a light-
directed
chemical process. The substrate may be paper, nylon or other type of membrane,
filter, chip, glass slide or any other suitable solid support.
In another aspect, an oligonucleotide may be synthesized on the surface of the
substrate by using a chemical coupling procedure and an ink jet application
apparatus,
as described in PCT application W095/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another aspect, a
"gridded" array
analogous to a dot (or slot) blot may be used to arrange and link cDNA
fragments or
oligonucleotides to the surface of a substrate using a vacuum system, thermal,
UV,
mechanical or chemical bonding procedures. An array, such as those described
above, may be produced by hand or by using available devices (slot blot or dot
blot
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apparatus), materials (any suitable solid support), and machines (including
robotic
instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more
oligonucleotides, or
any other number between two and one million which lends itself to the
efficient use
of commercially available instrumentation.
In order to conduct sample analysis using a microarray or detection kit, the
RNA or DNA from a biological sample is made into hybridization probes. The
mRNA is isolated, and cDNA is produced and used as a template to make
antisense
RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides,
and
labeled probes are incubated with the microarray or detection kit so that the
probe
sequences hybridize to complementary oligonucleotides of the microarray or
detection kit. Incubation conditions are adjusted so that hybridization occurs
with
precise complementary matches or with various degrees of less complementarity.
After removal of nonhybridized probes, a scanner is used to determine the
levels and
patterns of fluorescence. The scanned images are examined to determine degree
of
complementarity and the relative abundance of each oligonucleotide sequence on
the
microarray or detection kit. The biological samples may be obtained from any
bodily
fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured
cells,
biopsies, or other tissue preparations. A detection system may be used to
measure the
absence, presence, and amount of hybridization for all of the distinct
sequences
simultaneously. This data may be used for large-scale correlation studies on
the
sequences, expression patterns, mutations, variants, or polymorphisms among
samples.
Using such arrays, the present invention provides methods to identify the
expression of the transporter proteins/peptides of the present invention. In
detail, such
methods comprise incubating a test sample with one or more nucleic acid
molecules
and assaying for binding of the nucleic acid molecule with components within
the test
sample. Such assays will typically involve arrays comprising many genes, at
least
one of which is a gene of the present invention and or alleles of the
transporter gene
of the present invention. Figure 3 provides information on SNPs that have been
found
in the gene encoding the transporter protein of the present invention. SNPs
were
identified at 23 different nucleotide positions. Changes in the amino acid
sequence
caused by these SNPs can readily be determined using the universal genetic
code and
the protein sequence provided in Figure 2 as a reference. These SNPs may also
affect
control/regulatory elements.
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Conditions for incubating a nucleic acid molecule with a test sample vary.
Incubation conditions depend on the format employed in the assay, the
detection
methods employed, and the type and nature of the nucleic acid molecule used in
the
assay. One skilled in the art will recognize that any one of the commonly
available
5 hybridization, amplification or array assay formats can readily be adapted
to employ
the novel fragments of the Human genome disclosed herein. Examples of such
assays
can be found in Chard, T, An Introduction to Radioimmunoassay and Related
Technigues, Elsevier Science Publishers, Amsterdam, The Netherlands (1986);
Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press,
10 Orlando, FL Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P.,
Practice and
Theory of Enryme Immunoassays: Laboratory Techniques in Biochemistry and
Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands
(1985).
The test samples of the present invention include cells, protein or membrane
extracts of cells. The test sample used in the above-described method will
vary based
15 on the assay format, nature of the detection method and the tissues, cells
or extracts
used as the sample to be assayed. Methods for preparing nucleic acid extracts
or of
cells are well known in the art and can be readily be adapted in order to
obtain a
sample that is compatible with the system utilized.
In another embodiment of the present invention, kits are provided which
20 contain the necessary reagents to carry out the assays of the present
invention.
Specifically, the invention provides a compartmentalized kit to receive, in
close confinement, one or more containers which comprises: (a) a first
container
comprising one of the nucleic acid molecules that can bind to a fragment of
the
Human genome disclosed herein; and (b) one or more other containers comprising
25 one or more of the following: wash reagents, reagents capable of detecting
presence
of a bound nucleic acid.
In detail, a compartmentalized kit includes any kit in which reagents are
contained in separate containers. Such containers include small glass
containers,
plastic containers, strips of plastic, glass or paper, or arraying material
such as silica.
30 Such containers allows one to efficiently transfer reagents from one
compartment to
another compartment such that the samples and reagents are not cross-
contaminated,
and the agents or solutions of each container can be added in a quantitative
fashion
from one compartment to another. Such containers will include a container
which
will accept the test sample, a container which contains the nucleic acid
probe,
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51
containers which contain wash reagents (such as phosphate buffered saline,
Tris-
buffers, etc.), and containers which contain the reagents used to detect the
bound
probe. One skilled in the art will readily recognize that the previously
unidentified
transporter gene of the present invention can be routinely identified using
the
sequence information disclosed herein can be readily incorporated into one of
the
established kit formats which are well known in the art, particularly
expression arrays.
Vectors/host cells
The invention also provides vectors containing the nucleic acid molecules
described herein. The term "vector" refers to a vehicle, preferably a nucleic
acid
molecule, which can transport the nucleic acid molecules. When the vector is a
nucleic
acid molecule, the nucleic acid molecules are covalently linked to the vector
nucleic
acid. With this aspect of the invention, the vector includes a plasmid, single
or double
stranded phage, a single or double stranded RNA or DNA viral vector, or
artificial
chromosome, such as a BAC, PAC, YAC, OR MAC.
A vector can be maintained in the host cell as an extrachromosomal element
where it replicates and produces additional copies of the nucleic acid
molecules.
Alternatively, the vector may integrate into the host cell genome and produce
additional
copies of the nucleic acid molecules when the host cell replicates.
The invention provides vectors for the maintenance (cloning vectors) or
vectors
for expression (expression vectors) of the nucleic acid molecules. The vectors
can
function in procaryotic or eukaryotic cells or in both (shuttle vectors).
Expression vectors contain cis-acting regulatory regions that are operably
linked
in the vector to the nucleic acid molecules such that transcription of the
nucleic acid
molecules is allowed in a host cell. The nucleic acid molecules can be
introduced into
the host cell with a separate nucleic acid molecule capable of affecting
transcription.
Thus, the second nucleic acid molecule may provide a trans-acting factor
interacting
with the cis-regulatory control region to allow transcription of the nucleic
acid molecules
from the vector. Alternatively, a trans-acting factor may be supplied by the
host cell.
Finally, a trans-acting factor can be produced from the vector itself. It is
understood,
however, that in some embodiments, transcription and/or translation of the
nucleic acid
molecules can occur in a cell-free system.
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The regulatory sequence to which the nucleic acid molecules described herein
can be operably linked include promoters for directing mRNA transcription.
These
include, but are not limited to, the left promoter from bacteriophage 7~, the
lac, TRP, and
TAC promoters from E coli, the early and late promoters from SV40, the CMV
immediate early promoter, the adenovirus early and late promoters, and
retrovirus long-
terminal repeats.
In addition to control regions that promote transcription, expression vectors
may
also include regions that modulate transcription, such as repressor binding
sites and
enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate
early
enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR
enhancers.
In addition to containing sites for transcription initiation and control,
expression
vectors can also contain sequences necessary for transcription termination
and, in the
transcribed region a ribosome binding site for translation. Other regulatory
control
elements for expression include initiation and termination codons as well as
polyadenylation signals. The person of ordinary skill in the art would be
aware of the
numerous regulatory sequences that are useful in expression vectors. Such
regulatory
sequences are described, for example, in Sambrook et al., Molecular Cloning: A
Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, (1989).
A variety of expression vectors can be used to express a nucleic acid
molecule.
Such vectors include chromosomal, episomal, and virus-derived vectors, for
example
vectors derived from bacterial plasmids, from bacteriophage, from yeast
episomes, from
yeast chromosomal elements, including yeast artificial chromosomes, from
viruses such
as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses,
poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be
derived from
combinations of these sources such as those derived from plasmid and
bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning and
expression
vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al.,
Molecular
Cloning. A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, NY, (1989).
The regulatory sequence may provide constitutive expression in one or more
host
cells (i.e. tissue specific) or may provide for inducible expression in one or
more cell
types such as by temperature, nutrient additive, or exogenous factor such as a
hormone
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53
or other ligand. A variety of vectors providing for constitutive and inducible
expression
in prokaryotic and eukaryotic hosts are well known to those of ordinary skill
in the art.
The nucleic acid molecules can be inserted into the vector nucleic acid by
well-
known methodology. Generally, the DNA sequence that will ultimately be
expressed is
joined to an expression vector by cleaving the DNA sequence and the expression
vector
with one or more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well known to
those of
ordinary skill in the art.
The vector containing the appropriate nucleic acid molecule can be introduced
into an appropriate host cell for propagation or expression using well-known
techniques.
Bacterial cells include, but are not limited to, E coli, Streptomyces, and
Salmonella
typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect
cells such as
Drosophila, animal cells such as COS and CHO cells, and plant cells.
As described herein, it may be desirable to express the peptide as a fusion
protein. Accordingly, the invention provides fusion vectors that allow for the
production
of the peptides. Fusion vectors can increase the expression of a recombinant
protein,
increase the solubility of the recombinant protein, and aid in the
purification of the
protein by acting for example as a ligand for affinity purification. A
proteolytic cleavage
site may be introduced at the junction of the fusion moiety so that the
desired peptide can
ultimately be separated from the fusion moiety. Proteolytic enzymes include,
but are not
limited to, factor Xa, thrombin, and enterotransporter. Typical fusion
expression vectors
include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,
Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ) which fuse glutathione S-
transferase (GST), maltose E binding protein, or protein A, respectively, to
the target
recombinant protein. Examples of suitable inducible non-fusion E coli
expression
vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET l 1d
(Studier et
al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).
Recombinant protein expression can be maximized in host bacteria by providing
a genetic background wherein the host cell has an impaired capacity to
proteolytically
cleave the recombinant protein. (Gottesman, S., Gene Expression Technology:
Methods
in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
Alternatively, the sequence of the nucleic acid molecule of interest can be
altered to
provide preferential codon usage for a specific host cell, for example E coli.
(Wada et
al., Nucleic Acids Res. 20:2111-2118 (1992)).
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The nucleic acid molecules can also be expressed by expression vectors that
are
operative in yeast. Examples of vectors for expression in yeast e.g., S
cerevisiae include
pYepSecl (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al.,
Cell
30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2
(Invitrogen Corporation, San Diego, CA).
The nucleic acid molecules can also be expressed in insect cells using, for
example, baculovirus expression vectors. Baculovirus vectors available for
expression
of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al.,
Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al.,
Virology
170:31-39 (1989)).
In certain embodiments of the invention, the nucleic acid molecules described
herein are expressed in mammalian cells using mammalian expression vectors.
Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature
329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).
The expression vectors listed herein are provided by way of example only of
the
well-known vectors available to those of ordinary skill in the art that would
be useful to
express the nucleic acid molecules. The person of ordinary skill in the art
would be
aware of other vectors suitable for maintenance propagation or expression of
the nucleic
acid molecules described herein. These are found for example in Sambrook, J.,
Fritsh,
E. F., and Maniatis, T. Molecular Cloning.' A Laboratory Manual. 2nd, ed ,
Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1989.
The invention also encompasses vectors in which the nucleic acid sequences
described herein are cloned into the vector in reverse orientation, but
operably linked to a
regulatory sequence that permits transcription of antisense RNA. Thus, an
antisense
transcript can be produced to all, or to a portion, of the nucleic acid
molecule sequences
described herein, including both coding and non-coding regions. Expression of
this
antisense RNA is subject to each of the parameters described above in relation
to
expression of the sense RNA (regulatory sequences, constitutive or inducible
expression,
tissue-specific expression).
The invention also relates to recombinant host cells containing the vectors
described herein. Host cells therefore include prokaryotic cells, lower
eukaryotic cells
such as yeast, other eukaryotic cells such as insect cells, and higher
eukaryotic cells such
as mammalian cells.
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The recombinant host cells are prepared by introducing the vector constructs
described herein into the cells by techniques readily available to the person
of ordinary
skill in the art. These include, but are not limited to, calcium phosphate
transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated transfection,
5 electroporation, transduction, infection, lipofection, and other techniques
such as those
found in Sambrook, et al. (Molecular Cloning. A Laboratory Manual. 2nd, ed.,
Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
NY, 1989).
Host cells can contain more than one vector. Thus, different nucleotide
10 sequences can be introduced on different vectors of the same cell.
Similarly, the nucleic
acid molecules can be introduced either alone or with other nucleic acid
molecules that
are not related to the nucleic acid molecules such as those providing traps-
acting factors
for expression vectors. When more than one vector is introduced into a cell,
the vectors
can be introduced independently, co-introduced or joined to the nucleic acid
molecule
15 vector.
In the case of bacteriophage and viral vectors, these can be introduced into
cells
as packaged or encapsulated virus by standard procedures for infection and
transduction.
Viral vectors can be replication-competent or replication-defective. In the
case in which
viral replication is defective, replication will occur in host cells providing
functions that
20 complement the defects.
Vectors generally include selectable markers that enable the selection of the
subpopulation of cells that contain the recombinant vector constructs. The
marker can
be contained in the same vector that contains the nucleic acid molecules
described herein
or may be on a separate vector. Markers include tetracycline or ampicillin-
resistance
25 genes for prokaryotic host cells and dihydrofolate reductase or neomycin
resistance for
eukaryotic host cells. However, any marker that provides selection for a
phenotypic trait
will be effective.
While the mature proteins can be produced in bacteria, yeast, mammalian cells,
and other cells under the control of the appropriate regulatory sequences,
cell- free
30 transcription and translation systems can also be used to produce these
proteins using
RNA derived from the DNA constructs described herein.
Where secretion of the peptide is desired, which is difficult to achieve with
mufti-transmembrane domain containing proteins such as transporters,
appropriate
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56
secretion signals are incorporated into the vector. The signal sequence can be
endogenous to the peptides or heterologous to these peptides.
Where the peptide is not secreted into the medium, which is typically the case
with transporters, the protein can be isolated from the host cell by standard
disruption
procedures, including freeze thaw, sonication, mechanical disruption, use of
lysing
agents and the like. The peptide can then be recovered and purified by well-
known
purification methods including ammonium sulfate precipitation, acid
extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-
interaction chromatography, affinity chromatography, hydroxylapatite
chromatography,
lectin chromatography, or high performance liquid chromatography.
It is also understood that depending upon the host cell in recombinant
production
of the peptides described herein, the peptides can have various glycosylation
patterns,
depending upon the cell, or maybe non-glycosylated as when produced in
bacteria. In
addition, the peptides may include an initial modified methionine in some
cases as a
result of a host-mediated process.
s of vectors and host cells
The recombinant host cells expressing the peptides described herein have a
variety of uses. First, the cells are useful for producing a transporter
protein or peptide
that can be further purified to produce desired amounts of transporter protein
or
fragments. Thus, host cells containing expression vectors are useful for
peptide
production.
Host cells are also useful for conducting cell-based assays involving the
transporter protein or transporter protein fragments, such as those described
above as
well as other formats known in the art. Thus, a recombinant host cell
expressing a native
transporter protein is useful for assaying compounds that stimulate or inhibit
transporter
protein function.
Host cells are also useful for identifying transporter protein mutants in
which
these functions are affected. If the mutants naturally occur and give rise to
a pathology,
host cells containing the mutations are useful to assay compounds that have a
desired
effect on the mutant transporter protein (for example, stimulating or
inhibiting function)
which may not be indicated by their effect on the native transporter protein.
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Genetically engineered host cells can be further used to produce non-human
transgenic animals. A transgenic animal is preferably a mammal, for example a
rodent,
such as a rat or mouse, in which one or more of the cells of the animal
include a
transgene. A transgene is exogenous DNA that is integrated into the genome of
a cell
from which a transgenic animal develops and which remains in the genome of the
mature animal in one or more cell types or tissues of the transgenic animal.
These
animals are useful for studying the function of a transporter protein and
identifying and
evaluating modulators of transporter protein activity. Other examples of
transgenic
animals include non-human primates, sheep, dogs, cows, goats, chickens, and
amphibians.
A transgenic animal can be produced by introducing nucleic acid into the male
pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral
infection, and allowing
the oocyte to develop in a pseudopregnant female foster animal. Any of the
transporter
protein nucleotide sequences can be introduced as a transgene into the genome
of a non-
human animal, such as a mouse.
Any of the regulatory or other sequences useful in expression vectors can form
part of the transgenic sequence. This includes intronic sequences and
polyadenylation
signals, if not already included. A tissue-specific regulatory sequences) can
be operably
linked to the transgene to direct expression of the transporter protein to
particular cells.
Methods for generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become conventional in
the art
and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009,
both by
Leder et al., U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating
the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.,
1986). Similar methods are used for production of other transgenic animals. A
transgenic founder animal can be identified based upon the presence of the
transgene in
its genome and/or expression of transgenic mRNA in tissues or cells of the
animals. A
transgenic founder animal can then be used to breed additional animals
carrying the
transgene. Moreover, transgenic animals carrying a transgene can further be
bred to
other transgenic animals carrying other transgenes. A transgenic animal also
includes
animals in which the entire animal or tissues in the animal have been produced
using the
homologously recombinant host cells described herein.
In another embodiment, transgenic non-human animals can be produced which
contain selected systems that allow for regulated expression of the transgene.
One
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example of such a system is the crelloxP recombinase system of bacteriophage P
1. For
a description of the crelloxP recombinase system, see, e.g., Lakso et al. PNAS
89:6232-
6236 (1992). Another example of a recombinase system is the FLP recombinase
system
of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a crelloxP
recombinase system is used to regulate expression of the transgene, animals
containing
transgenes encoding both the Cre recombinase and a selected protein is
required. Such
animals can be provided through the construction of "double" transgenic
animals, e.g.,
by mating two transgenic animals, one containing a transgene encoding a
selected
protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced according to the methods described in Wilmut, I. et al. Nature
385:810-813
(1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In
brief, a cell, e.g., a somatic cell, from the transgenic animal can be
isolated and induced
to exit the growth cycle and enter Go phase. The quiescent cell can then be
fused, e.g.,
through the use of electrical pulses, to an enucleated oocyte from an animal
of the same
species from which the quiescent cell is isolated. The reconstructed oocyte is
then
cultured such that it develops to morula or blastocyst and then transferred to
pseudopregnant female foster animal. The offspring born of this female foster
animal
will be a clone of the animal from which the cell, e.g., the somatic cell, is
isolated.
Transgenic animals containing recombinant cells that express the peptides
described herein are useful to conduct the assays described herein in an in
vivo context.
Accordingly, the various physiological factors that are present in vivo and
that could
effect ligand binding, transporter protein activation, and signal
transduction, may not be
evident from in vitro cell-free or cell-based assays. Accordingly, it is
useful to provide
non-human transgenic animals to assay in vivo transporter protein function,
including
ligand interaction, the effect of specific mutant transporter proteins on
transporter protein
function and ligand interaction, and the effect of chimeric transporter
proteins. It is also
possible to assess the effect of null mutations, that is mutations that
substantially or
completely eliminate one or more transporter protein functions.
All publications and patents mentioned in the above specification are herein
incorporated by reference. Various modifications and variations of the
described
method and system of the invention will be apparent to those skilled in the
art without
departing from the scope and spirit of the invention. Although the invention
has been
described in connection with specific preferred embodiments, it should be
understood
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that the invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the above-described modes for
carrying out the invention which are obvious to those skilled in the field of
molecular
biology or related fields are intended to be within the scope of the following
claims.
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SEQUENCE LISTING
<110> PE CORPORATION (NY)
<120> ISOLATED HUMAN TRANSPORTER PROTEINS,
NUCLEIC ACID MOLECULES ENCODING HUMAN TRANSPORTER PROTEINS,
AND USES THEREOF
<130> CL001065PCT
<140> TO BE ASSIGNED
<141> 2002-O1-02
<150> 09/752,821
<151> 2001-O1-03
<150> 09/861,846
<151> 2001-05-22
<160> 4
<170> FastSEQ for Windows Version 4.0
<210> 1
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<213> Homo sapiens
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atggctcatg ccccagaacc agacccggcc gccagcgacc tcggggatga gaggcccaag 60
tgggacaaca aggcccagta cctcctgagc tgcatcgggt ttgccgtggg gctggggaac 120
atttggcggt tcccatacct gtgccagacc tatggaggag gggccttcct catcccctac 180
gtcatcgcgc tggtcttcga ggggatcccc attttccacg tcgagctcgc catcggccag 240
cggctgcgga agggcagcgt cggcgtgtgg acggccatct ccccgtacct cagtggagta 300
gggctgggct gtgtcacgct gtccttcctg atcagcctgt actacaacac catcgtggcg 360
tgggtgctgt ggtacctcct caactccttc cagcacccgc tgccctggag ctcctgccca 420
ccggacctca acagaacagg gtttgtggag gagtgccagg gcagcagcgc cgtgagctac 480
ttctggtacc ggcagacact gaacatcaca gccgacatca atgacagtgg ctccatccag 540
tggtggctgc tcatctgctt ggcggcctcc tgggcagtcg tgtacatgtg tgtcatcagg 600
ggcattgaga ctacagggaa ggtgatttac ttcacagctt tgttccctta cctggtcctg 660
accatctttc tcatcagagg gctgaccctg ccaggggcaa caaaaggact catctacttg 720
ttcactccca acatgcacat tctccaaaac ccccgggtgt ggctggacgc agccacccag 780
atattcttct ctctgtccct ggccttcgga ggacacatcg cttttgcaag ttacaactcg 840
cccaggaatg actgccagaa ggatgcggtg gtcatcgccc tggtcaacag gatgacctcc 900
ctgtacgcgt ccatcgctgt cttctctgtc ctggggttca aagcaactaa tgactacgag 960
cactgcctgg acagaaacat cctcagcctc atcaacgact ttgacttccc agagcagagc 1020
atctccaggg acgactaccc agccgtcctc atgcacctga acgccacctg gcccaagagg 1080
gtggcccagc tccccctgaa ggcctgcctc ctggaagact ttctggataa gagtgcctcg 1140
ggcccgggcc tggccttcgt cgtcttcacg gagaccgacc tccacatgcc gggggctcct 1200
gtgtgggcca tgctcttctt cgggatgctg ttcaccttgg ggctatcgac catgttcggg 1260
accgtggagg cggtcatcac acccctgctg gacgtggggg tcctgcctag atgggtcccc 1320
aaggaggccc tgactgggct ggtctgcctg gtctgcttcc tctccgccac ctgcttcacg 1380
ctgcagtctg ggaactactg gctggagatt ttcgacaatt ttgccgcttc cccgaacctg 1440
ctcatgttgg cctttctcga ggttgtgggt gtcgtttatg tttatggaat gaaacggttc 1500
tgcgatgaca ttgcgtggat gaccgggagg cggcccagcc cctactggcg gctgacctgg 1560
agggtggtca gtcccctgct gctgaccatc tttgtggctt acatcatcct cctgttctgg 1620
aagccactga gatacaaggc ctggaacccc aaatacgagc tgttcccctc gcgtcaggag 1680
aagctctacc cgggctgggc gcgcgccgcc tgtgtgctgc tgtccttgct gcccgtgctg 1740
tgggtcccgg tggccgcgct tgctcagctg ctcacccggc ggaggcggac gtggagggac 1800
agggacgcgc gcccagacac ggacatgcgc tga 1833
<210> 2
<211> 610
<212> PRT
<213> Homo sapiens
<400> 2
CA 02433579 2003-07-02
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Met Ala His Ala Pro Glu Pro Asp Pro Ala Ala Ser Asp Leu Gly Asp
1 5 10 15
Glu Arg Pro Lys Trp Asp Asn Lys Ala Gln Tyr Leu Leu Ser Cys Ile
20 25 30
Gly Phe Ala Val Gly Leu Gly Asn Ile Trp Arg Phe Pro Tyr Leu Cys
35 40 45
Gln Thr Tyr Gly Gly Gly Ala Phe Leu Ile Pro Tyr Val Ile Ala Leu
50 55 60
Val Phe Glu Gly Ile Pro Ile Phe His Val Glu Leu Ala Ile Gly Gln
65 70 75 80
Arg Leu Arg Lys Gly Ser Val Gly Val Trp Thr Ala Ile Ser Pro Tyr
85 90 95
Leu Ser Gly Val Gly Leu Gly Cys Val Thr Leu Ser Phe Leu Ile Ser
100 105 110
Leu Tyr Tyr Asn Thr Ile Val Ala Trp Val Leu Trp Tyr Leu Leu Asn
115 120 125
Ser Phe Gln His Pro Leu Pro Trp Ser Ser Cys Pro Pro Asp Leu Asn
130 135 140
Arg Thr Gly Phe Val Glu Glu Cys Gln Gly Ser Ser Ala Val Ser Tyr
145 150 155 160
Phe Trp Tyr Arg Gln Thr Leu Asn Ile Thr Ala Asp Ile Asn Asp Ser
165 170 175
Gly Ser Ile Gln Trp Trp Leu Leu Ile Cys Leu Ala Ala Ser Trp Ala
180 185 190
Val Val Tyr Met Cys Val Ile Arg Gly Ile Glu Thr Thr Gly Lys Val
195 200 205
Ile Tyr Phe Thr Ala Leu Phe Pro Tyr Leu Val Leu Thr Ile Phe Leu
210 215 220
Ile Arg Gly Leu Thr Leu Pro Gly Ala Thr Lys Gly Leu Ile Tyr Leu
225 230 235 240
Phe Thr Pro Asn Met His Ile Leu Gln Asn Pro Arg Val Trp Leu Asp
245 250 255
Ala Ala Thr Gln Ile Phe Phe Ser Leu Ser Leu Ala Phe Gly Gly His
260 265 270
Ile Ala Phe Ala Ser Tyr Asn Ser Pro Arg Asn Asp Cys Gln Lys Asp
275 280 285
Ala Val Val Ile Ala Leu Val Asn Arg Met Thr Ser Leu Tyr Ala Ser
290 295 300
Ile Ala Val Phe Ser Val Leu Gly Phe Lys Ala Thr Asn Asp Tyr Glu
305 310 315 320
His Cys Leu Asp Arg Asn Ile Leu Ser Leu Ile Asn Asp Phe Asp Phe
325 330 335
Pro Glu Gln Ser Ile Ser Arg Asp Asp Tyr Pro Ala Val Leu Met His
340 345 350
Leu Asn Ala Thr Trp Pro Lys Arg Val Ala Gln Leu Pro Leu Lys Ala
355 360 365
Cys Leu Leu Glu Asp Phe Leu Asp Lys Ser Ala Ser Gly Pro Gly Leu
370 375 380
Ala Phe Val Val Phe Thr Glu Thr Asp Leu His Met Pro Gly Ala Pro
385 390 395 400
Val Trp Ala Met Leu Phe Phe Gly Met Leu Phe Thr Leu Gly Leu Ser
405 910 415
Thr Met Phe Gly Thr Val Glu Ala Val Ile Thr Pro Leu Leu Asp Val
420 425 930
Gly Val Leu Pro Arg Trp Val Pro Lys Glu Ala Leu Thr Gly Leu Val
435 440 945
Cys Leu Val Cys Phe Leu Ser Ala Thr Cys Phe Thr Leu Gln Ser Gly
450 455 960
Asn Tyr Trp Leu Glu Ile Phe Asp Asn Phe Ala Ala Ser Pro Asn Leu
465 470 475 480
Leu Met Leu Ala Phe Leu Glu Val Val Gly Val Val Tyr Val Tyr Gly
485 990 495
Met Lys Arg Phe Cys Asp Asp Ile Ala Trp Met Thr Gly Arg Arg Pro
500 505 510
Ser Pro Tyr Trp Arg Leu Thr Trp Arg Val Val Ser Pro Leu Leu Leu
515 520 525
Thr Ile Phe Val Ala Tyr Ile Ile Leu Leu Phe Trp Lys Pro Leu Arg
530 535 540
2
CA 02433579 2003-07-02
WO 02/053741 PCT/US02/00111
Tyr Lys Ala Trp Asn Pro Lys Tyr Glu Leu Phe Pro Ser Arg Gln Glu
545 550 555 560
Lys Leu Tyr Pro Gly Trp Ala Arg Ala Ala Cys Val Leu Leu Ser Leu
565 570 575
Leu Pro Val Leu Trp Val Pro Val Ala Ala Leu Ala Gln Leu Leu Thr
580 585 590
Arg Arg Arg Arg Thr Trp Arg Asp Arg Asp Ala Arg Pro Asp Thr Asp
595 600 605
Met Arg
610
<210> 3
<211> 13608
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<222> (1). .(13608)
<223> n = A,T,C or G
<400> 3
attaaactgg tgttctgaga gttcctacgg acaggtcacc tccgctttcc atagaagaat 60
ctggacgctt acattaaact gatgttctga gaattcctac aggcaggact gaaagcctgg 120
tgtgtgccag tatgatgttc cacccacgga aacctggtca caatcgtccc ttccagcacc 180
ccatccagca gtgactgcac acactgagcc tcctaccagc ccctttcacc ctgctgactg 240
tcactgggcc cctgggatgt gcaagactcc acagcagcag aggtgggggg acatatcaca 300
gcctctgccc ccggctgtga tgccaccgag gggctcgcct gctgatggct tcaacagggt 360
ctcacctcat cttttcctgc tctttggccc tggatcgaga aaatttccat cagtgcccca 420
ttaatatgct gccctgtggc atctgcccag gaggccctgc caggcgtgca caggtgtgca 480
ttggtgtacc ctggcatgca caggtgtgca ctgatgtgcc ctggcatcca ttggtgtacc 540
ctggtgtgcc tgccatagga ccctgggcgg gagctcccat ctcatctaca tctcctgatt 600
catgcgttgt ttcataggtt tcaatgtctc tgtaaatgtg gtagaaatgc aggctttatg 660
ggcataaagt gtacatttct aaataaatcc cttctattta gtatgctcac cctagaagtt 720
actgttgtcc agacgtagag ggatgagtga gccagtgacc tcagacggga tggtggggac 780
ggcaggtcca gctcctgcct cctcctgggg ggtctggctt tgggggcttg ctccgaagag 840
gccatggccc aggcctgtgg cctcacaatg gggaccaacc agctcttctc atcttcttcc 900
ctcacacttc ctctcactca aataagaacc ttccaaaaat gtgtccacct gggcccctgc 960
cctgggactc atggatttgg agttgtggcc acacggttga gggctgcagt gtccagtgga 1020
atggggcaat tgcgggcctg ggggcccttg gcctgtccgt ggcgggagca tctgcaagga 1080
ggagccccag agtccaggga gcactgtggg gagctcctta gagctgaact cacccggcgt 1140
caactcatca accctccacc catggacagg ggtgccccca gcacaggaga ggactcagcc 1200
ctctgccccc acgcacggtg ggtgcctgtc accctgtcct gcccagcggc ccgagggcag 1260
cagtgggtgt gagggcagcc cccggcctcc caagagcagc tgagaggatc cctgcgggaa 1320
tccgggcttt gggtgcatgc gatctgatct gagttgtttc tgacagtgac agagtgacaa 1380
tctataagta tctcaagatc aaatggttaa ataaaacata agaaatttaa aacgattaaa 1440
atatgcctca gtcttgggaa tcctggtgtc tgcaacaaaa aaacttgagg ctgtccttgc 1500
aaacggtcct catggtgctg ggggtggtgc caggcccggc gtttgcctgg aacacagagc 1560
atgcgagaag gagcagagga cgtgggtgcc agggaaggac cctggcgtca gagccagggc 1620
ctggacctga gttacccatt aatggcactc cactggtttt cctttaaaga aatgaataaa 1680
tacaactgca gtggggctgc ttgtggtttc caaacgtcgg cagaggctgg agacggctct 1740
ctagtgctgg gtgtggagtg aggcaccacc ctcgccctga agcctggggc actcagtcac 1800
catggctcat gccccagaac cagacccggc cgccagcgac ctcggggatg agaggcccaa 1860
gtgggacaac aaggcccagt acctcctgag ctgcatcggg tttgccgtgg ggctggggaa 1920
catttggcgg ttcccatacc tgtgccagac ctatggagga ggtaagcacc cacctgcgtc 1980
ctggggcaga cccctaagca ggggaggcca tctccaccct gacctgccag gcgcctgcca 2040
cgcatcccca gtgcttgggc agagtcactc ctcctggaaa ccagggtgca ccattgccca 2100
cggcagagtc agtcccctca gggcccacct gcagtcttct gggacctcag ggcagcttca 2160
ccactgccca cagcacggag gatccagtcc cctcggggtc cactatggtc ttccgggacc 2220
tcagggcagc ttcctgccag gcgtcaggtt gaatgggcca ccctgcccga cacacacacc 2280
ctacaaacag tgagcgctct tccatctgaa tgtcgtcagt tcttttaaga acacacaatc 2340
cgctctgggt ttcctcacca cagccttggt gtgtccagac atgtgctcag gtgcccttct 2900
ccgaggttct ttgcaggaat ttgggggccc acccctcgcc tccctggctt tggttctctg 2960
ggagagccct gtggggggtg ccaggatgtt cagaactgtc tgggtgactt tagtgacctt 2520
ggactcgcca cccccagaag tgcattggtg actgaaatag gctcccacgc accccgagcc 2580
tcccctcacc ctgtgcctgc ttctcttcat gcaggggtca cactctgctc tccatgtggc 2640
gacacaggat gtagggacag gcatgaatca ctgctctcag ggagccgacg tacaacatca 2700
CA 02433579 2003-07-02
WO 02/053741 PCT/US02/00111
cgcatggaca cccaggtctt ccccaggcaa cacgggcagg ggcttcccag gccgcagggt 2760
cctcctcagg ccaggccggg ctgcttggtc agcctgttcc ccgggccctc agctccttcc 2820
ctgagagccc cgcacaacta tcccttgtta taaggttact cctttaaagt gtttttcctc 2880
ctgcttaaga aaagattctc aacaggcaga aaatgcagga aatgcgcaca aatggaaaaa 2940
acaaaaagaa acacccgtca ctgccaccct cggggcactg gggtctcttt ggggctttgg 3000
ggctggagag ggtgcattgg gccttggatc ttctcatcat gagagtccct ttgaaggcat 3060
ttctgaaagc accaaagccc aaggacagag gcgtccctgg ctcccggggc cctcgctgcc 3120
tgtgctgcgg ctcgatctgc taagagtcac cagagcatgc aaggttgtgg gggcagcagg 3180
cttcacgcag agctggaaat gccggccaac gctttaccca ccgacgcctt gttcgccaat 3240
gccttgccca ccgatgcctt tcctgctgat gccttgccca acgccttgcc caccgacgcc 3300
ttgcccaccg atgccttgcc caccgatnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3360
nnnnnnnnnn nnnnnattca catgggagcc tggccctgag aaaggcaggt atgctgactc 3420
cctgccatga gctccctggg gtgccctggg ttttgttccc ttttgctcca ggccctggcc 3480
acagctgttc ccaccctccc aggaggcatc ttggggtgtc aggtgcctcc acctgaggct 3540
gaccccgtag acctcagctt cccctcttca atgcttgtct ttctgccgct gtgtaaatga 3600
cacggacaag agccgtccgg cccccacatg ctggccctgg agtcctgtgc agaagccaac 3660
aggggtgagg gctcacctcc cactctaggg cctccctgtg cccgtggcag tcgcagaaca 3720
ttctcatggt gacacaaacc cagagcaagg ctgtgttgat gacctggaaa cgagcttcta 3780
gaagccggac cctgcctggc aaagtggagc tgatgggaga tgccaggcgc ggagacgcac 3840
ggcggggaag agtgtgggca gccggcaccc atgtaggtca gcgtcttagg gcagaaggcc 3900
tgaggtcagt gccaccccgc agcctgtccc agcacctgtg catgcaggtg gggaggagtc 3960
aacagccctg ggcacagacg tggagctgca cagagacccg aactcgtgca cagacggtga 9020
ctcgcttggg gaacagtgca gctgccaact ctgtgaggcc accaggactg agacgcagca 4080
tgcagacaca ggggcctctt cagcagcgta gtgatgccca tggaaccagg agcccgggag 4140
aaccagcatc ctcccagcag gtcttgtagg cagctgctga acttagaggt caaattgcag 4200
gaatcgatca tgcccagctc tgccgctggg gccaggaggg tctgtggact ccacgggaag 4260
ccagccctgc tcgcatcctc agcaccaggc cctggcacct tgtctgcccc ttccactgcg 4320
ggtgccccag gcagaaggca cggccattca tctctactgg gtgcttccac tagctccagg 4380
aagtggagcc ggcctgttcc ccctgccctg gggcctgtgg ggccttcatc ctgccatgcc 4440
tatgggcacc cccatcatgg cactggatgg aggtacagag gcccgagatg ccaggaagtg 4500
gggggactgg tccagccctg atgcagctct cagagccttg gtctgagaca ccactctcct 4560
gcgacatccc catgagcaaa gatggttcca gttcccagga gagaccgcca gccgccccgg 4620
gttcctcgtg ggaacagagc agccctcaag ccgccctggg gtccctgggg gaacagagca 4680
gctcatgcca tctgttgcat gaagctccaa caagagcagc atgaagccgg ggagccaaca 4740
ggcgacctca cacaaaagca gccagacctc ctccggtgac tggggtgttt aaaaaacatg 4800
ctcagtcagc actttatttc tgcagactca tgaattttca gaaacatcac gttaatgaaa 4860
ggagctgttt tcccatgggg aggctgatgg cagaggtttt cagtgtgatt ccgccaggcc 4920
cctgcaggcc tccttcccac agaggctgag tccccactgc cggccacagc agcccccagg 4980
aaaaggcagg tgcctcaaac aatgctggtc cctcagcaaa cgcaacacag gggtcacgct 5040
tagaccccaa gtcctagtcc agggccggct gcctcctttt gggccccctg ctcccctcca 5100
gggccctgcc ctcttgatga gaggtctcag caaccgagcc aaaatcagag gcagggtttg 5160
gcaacccaac agtgccccaa gggtgtctcc accacccaag tggtgccccc aacattcagg 5220
tcccgtctgc cccttgaaga ccacaggtgg ctccccctgg cccacgccac agtccccccc 5280
agcccccgac cctgggcagg tgctgaccac cccctcctca caggggcctt cctcatcccc 5340
tacgtcatcg cgctggtctt cgaggggatc cccattttcc acgtcgagct cgccatcggc 5400
cagcggctgc ggaagggcag cgtcggcgtg tggacggcca tctccccgta cctcagtgga 5460
gtaggtaggc caccgtcctc ccttgccctg actgaggctg ccagggacag ggccctcctg 5520
gatgagaggt ggggcggggg cgggtccatg cctgtggtac ggaagcggcc aggccaggcc 5580
ggcgggtggg gtggcaggga gcccttgggt gtgtgtgaga agcagcggtg actcggggag 5640
aattagagat ggagaccatg tgtgccagga catcccggaa ggacctggaa gctggtgttg 5700
ccattcacat gtgaggtgtg agggaggcct ggctttcagc tgcgcccttc agcatgtgtt 5760
atttaatgtt gttattttgt tttattctca ctgcttctgg gcagagggag ctgggaagga 5820
gccccggggc cacctgacat ggtccctgtc cacagggctg ggctgtgtca cgctgtcctt 5880
cctgatcagc ctgtactaca acaccatcgt ggcgtgggtg ctgtggtacc tcctcaactc 5940
cttccagcac ccgctgccct ggagctcctg cccaccggac ctcaacagaa caggtgagct 6000
gggcgccgcc tgctgtgtgg gtccgtgcac ggccgagaga ggcatgtgct gcagcgtgtc 6060
cagcatcaga gcagctgcgg gtggcggatg ctcaccgcgg ggggagggcc ggggaaccgg 6120
ttgctctgtg tgcacatgca cgcgcctcgg tctccccagg agcacacgtg cagtccagtt 6180
cttatgacct ccacgctgtg ctgtgcttcc ctgccaggtc cggaccaccg aggtcccctt 6240
gcagccatgt gcatggcgtg gtcatgcgag ggcactgctg ttgttagatt taagacgtta 6300
ggtcggacgc cgtggctcac gcttgtcaac ccagcacttt gggaggccga ggtgggcaga 6360
tcacttgagg tcaggagttc aagaccagcc tgaccaatat ggtgaaaccc catctctact 6420
caaaatacaa aaatcagcca ggcatggtgg cgggcatctg taatcccagc tagccaggag 6480
gctgagacag aagaattgct tgaacccggg aagtggaggt tgcagatagc cgagatcata 6540
ccactgcact ccagacgggg tgacagagtg agactctgta tcaaaaaaca atcgaacaaa 6600
caaannnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntggtgcaa 6660
gcatggctca ctgcaacctc caactcctgg ctcaagtgat tctcctacct ccacttccca 6720
agcagctggg accacagaca agtgcctcca cactcagcta attttttttt tttttttttt 6780
4
CA 02433579 2003-07-02
WO 02/053741 PCT/US02/00111
gagacggagt cttgctctgt cgcccaggct ggagtgcagt ggcgctatct tggctcactg 6840
caagctctgc ctcccaggtt cacgccattc tcctgcctca gcctcccaag tagctaggac 6900
tacaggcgcc tgccaccagg cctggctaat tttttgtatt tttagtagag acggggtttc 6960
accatgttag ccaggatggt ctcgatctcc tgacctcgtg atccgcctgc ctcggcctcc 7020
caaagtgctg ggattacagg cgtgagccac cgcgcccggc cagcacccag ctaattttat 7080
tttttgtaga gacggggtct caatatgttg cccaggatga tcttgaaccc ttgggttcaa 7140
gcgatcctcc tgcttcaggc tcccaaagca ctgggatgac aggtgtgagc cactgcactc 7200
aactgacagt cgtatctttc aatgacttgc ttaggaagca actctgactt tctttccttc 7260
ctgccttttg ctaaaactca cgctgggctg gtgctggatg aggctggcct cagggacgaa 7320
cactgagtct gcacctggat gccctgggcc agagctgggc accctgggga ggccaagagc 7380
cctggctgtg ccatcagcct cctggcacct ctctgagggt gggtggtcct gccagggttc 7440
ctcccagcca cactccagaa gcggccccag tacaggctat ctgcagcccg agttcctggg 7500
tgatggtttg tgggtcccaa attccttcta ccttgtccaa gcgtttactt ttggggtcac 7560
ttaggccagc cctttgattt tcaggcaggc cgagaggagt taagtcctct ctcaatgcac 7620
acggcagggc cccataactg cccttcccac atctgcccca agctccactg aaggaaaatt 7680
tccgaagaaa agggcccgga ggggccagct ctgcccatgc ccttccctca gccaagcgtg 7740
gggcaggcag tgcccactca gagctgggct gtgacagtgc ttgatgtcaa tgactggcag 7800
ggccttagaa ggagacctag aagaagcctt ggggcccaag gacagtgtgg agggaggagc 7860
cctgtggcat gggcagtggc ccggtcggga taccggtaat cctcagactt ttccatgagg 7920
ggccccccac atttccccag tcaagtgtcc cagaaatggg ctgcagtggg tcctgagaga 7980
tgtcacggga gcctgctgtt gtcagacgct gcctctggat ccctgcatca gcctgtggcc 8040
ccgtcaagga ggaagtggga gtcacaggac aatctgcccg cctggccaca gcctcacttt 8100
ctccccaagc actagccagc cacacacttg acacatgcag ccgctctccg tccagtgctc 8160
ctgtgccatc cttgggggtc tgggatggtg ggtggcagga ggaagtgagg ggaccagctt 8220
tacatggtga cagtgacgtc caaggggctg caggctgccc cggctcagca gggctcgtaa 8280
accttcgcaa agtggagctt ccctttgagg ttcactcagc ctgttcattt tcacagccac 8340
gtctgtttct ccatctcagc cctgggccca aaaagaagtg aaagggcacc tcagcccaaa 8400
agggcagtgt tgtgagcagc ccagggaaat tgagctcaag agccacatgg tgagggtgcc 8460
tacgccccac agccagcctg tgacagg.ccc cctggtttca gggtttgtgg aggagtgcca 8520
gggcagcagc gccgtgagct acttctggta ccggcagaca ctgaacatca cagccgacat 8580
caatgacagt ggctccatcc agtggtggct gctcatctgc ttggcagcct cctgggcagt 8640
cgtgtacatg tgtgtcatca ggggcattga gactacaggg aaggtgagag ctggcagggc 8700
ctgatcccct ctcttgcttc ctccagcccc caagacccct ccccattgac ctgtgttcgc 8760
tttgtgtcta caagctcttg cgaggggcat aaacatctag gattgcaacg tcttcttagg 8820
gaattgatcc cattatcact gtgaggcaac cttttttatt ctggtgatat tcttggctct 8880
gaaactgact ttgttttaaa atagccactc cagatttctt tgggctatgt taacagagtg 8940
tattttttcc atcctttttc ttttaaactt tttgtatctt tatgttgaaa gtgtttttct 9000
tgtagacagc atacaattgg atctggctgt tttaaccaat ctgacaatct ctgcctttta 9060
tttggggtgt ttagagcatt tacatttaat gtgattattg atatggttgg gtttagcgct 9120
gtcctcagat gtgtttccat gtgccccatc tgtttttttt ttattcttgt ttctctcttt 9180
ctctaggtat ttttcaggat cccgctctat ctccttggtt ggtttagctt taactctctc 9240
ttgttattct agtaactgct ttagaggtta cagtctacat ctctagcctg tcatggtcta 9300
tcattaagtg atcttggggc attctatggt catttaagaa tctcattgca ttttatccca 9360
tttcccttcc caggctttgc gctattgttg tcatacattt tgcttataac atgttatata 9420
aacagcaaat gcattgttat cagtttagtt ttaaacaagc gattatcttt caaagaggct 9480
taaataatga gaaaaactgt attgatgttc aaccatgtgg tttccattgg gagcagcaga 9540
aaggaccagt agaacctgag cctcctgctt gcctggaccc ctcaagcaag aagagtcctt 9600
accctgtatt ctctagtgtg tgctatcatc tccagtctgg tatcacacat gtgtggattc 9660
ttttcaaatg acggtgctca gtccatgcac cctggaccgt gcctcctgac ccatgggcca 9720
cagacacagg accccagtgc cccatgcgct gtgcagtgtc tcaggacgcc agtgccccat 9780
gtcctgtgca gtgtctcagg acgccagtgc cccatgccct gtgcagtgtc tcagtgtgct 9840
gaggttgtca ttcctcttac atggagggtg ggggtcttca tgcttgagag gactggaatt 9900
actgaattag acaaagcagc cagccagaaa ttccaaataa tcctattata aaggccctta 9960
aaaatgcatc ccaagctggg cacagtggct catgcctgta atcccagcac tttgggaggc 10020
taaggtaggt ggatcacatg aagtcaggag ttcaagacca gcctcgccaa catagtgaca 10080
ccccgtgtct actaaaaata aaaaaaaaaa aaaattagcc agccgtggtg ttgggagcct 10140
gtaatcccaa ctacttgcga ggctgaggca gaagaattgc ttgaacccgg aagcagnnnn 10200
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngcagga ggcgtccaca 10260
cgatcccaac aggggcaggt aggcgtccac acgatcccaa caggggcagg gaggcgtcca 10320
cacgatccca acaggggcag gaggcgtcca cacgatccca acaggggcag gaggggccca 10380
cacgatccca acaggggcag gaggggccca gccctcccag ggatgctgtg cttcgcagca 10940
ccaaccaagg gtttctctgg atgtgtttgg gaaccagggc catgagccca cagtcctctc 10500
tgtccccgca gaaacatcct cagcctcatc aacgactttg acttcccaga gcagagcatc 10560
tccagggacg actacccagc cgtcctcatg cacctgaacg ccacctggcc caagagggtg 10620
gcccagctcc ccctgaaggc ctgcctcctg gaagactttc tggataaggt acctgcacac 10680
cccctggggt agccaggcgg ggccgtccac aggagcacca gggccgcact ggctgtgtcc 10740
cactgccaaa ggctgcaaac aattcccaca gacccagggc cagggcctgt gaaccaagaa 10800
ccccagtcta tctttgtctc aggttgccag gcctcctgga aagcccgaag tcctgggggc 10860
CA 02433579 2003-07-02
WO 02/053741 PCT/US02/00111
agctgtgtcc agcagacgct gcacccagca gtcttggggg ttgggctgac ccgagagagt 10920
gggcattgct ggtgcccaga ccccaggaga gggggtggcc agcatgaggg tcacagtgcc 10980
agggcatggc tggggtcaag ccacactcag ggccagtgta tgcctggacc taggggcacc 11040
aggcaggggg gcacagccag gaggcaggtg tgggatgcat ctcagggtgc ctgactctag 11100
gcataaaagg cgctggtttc taggcccagc tgcctgctgt gggttattaa aggggaagga 11160
ccctcccctg cagagttgcg cttgcagcct cgtctcccag aaggcatggc cagccctgag 11220
ccacctcagc ccgacaccag gaggggtgat gtgcactcgt gtcctcggcc tgggagagtg 11280
tgtgtcctgc aggcaggcgt gtgtgtgtgg tggagtgtgt gtgtgcgtgg cctgaagccc 11340
ggggctccgt gtattgcaga gtgcctcggg cccgggcctg gccttcgtcg tcttcacgga 11900
gaccgacctc cacatgccgg gggctcctgt gtgggccatg ctcttcttcg ggatgctgtt 11460
caccttgggg ctatcgacca tgttcgggac cgtggaggcg gtcatcacac ccctgctgga 11520
cgtgggggtc ctgcctagat gggtccccaa ggaggccctg actggtgagc gcacagctcc 11580
gccgccctgg aggacccgtc cccagcatct gactgtccac tcccgcccgc tgtccagacg 11640
cccctcctgg atggagagcg caaggggcca agcctgagtt cagggaaagg ctgagccagg 11700
ctactccttg ctgacagcca tcaacggaga gccgagggtc gagggagggt cagggctgcc 11760
cctcccccac ggccccggag gccacatccc ccatctggag caggaaagca ggtccttcct 11820
gctccttgag cacaatacag gaaagagact ggggagggca gggatggagg gtgggaagcc 11880
gagagaagtc tccagcccag gtnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11940
nnnnnnnnnn acctgggctg gagacttctc tcggcttccc accctccatc cctgccctcc 12000
ccagtctctt tcctgtattg tgctcaagga gcaggaagga cctgctttcc tgctccagat 12060
gggggatgtg gcctccgggg ccgtggggga ggggcagccc tgaccctccc tcgaccctcg 12120
gctctccgtt gatggctgtc agcaaggagt agcctggctc agcctttccc tgaactcagg 12180
cttggcccct tgcgctctcc atccaggagg ggcgtctgga cagcgggcgg gagtggacag 12290
tcagatgctg gggacgggtc ctccagggcg gcggagctgt gcgctcacca gtcagggcct 12300
ccttggggac ccatctaggc aggaccccca cgtccagcag gggtgtgatg accgcctcca 12360
cggtcccgaa catggtcgat agccccaagg tgaacagcat cccgaagaag agcatggccc 12420
acacaggagc ccccggcatg tggaggtcgg tctccgtgaa gacgacgaag gccaggcccg 12480
ggcccgaggc actctgcaat acacggagcc ccgggcttca ggccacgcac acacacactc 12540
caccacacac acacgcctgc ctgcaggaca cacactctcc caggccgagg acacgagtgc 12600
acatcacccc tcctggtgtc gggctgaggt ggctcagggc tggccatgcc ttctgggaga 12660
cgaggctgca agcgcaactc tgcaggggag ggtccttccc ctttaataac ccacagcagg 12720
cagctgggcc tagaaaccag cgccttttat gcctagagtc aggcaccctg agatgcatcc 12780
cacacctgcc tcctggctgt gcccccctgc ctggtgcccc taggtccagg catacactgg 12840
ccctgagtgt ggcttgaccc cagccatgcc ctggcactgt gaccctcatg ctggccaccc 12900
cctctcctgg ggtctgggca ccagcaatgc ccactctctc gggtcagccc aacccccaag 12960
actgctgggt gcagcgtctg ctggacacag ctgcccccag gacttcgggc tttccaggag 13020
gcctggcaac ctgagacaaa gatagactgg ggttcttggt tcacaggccc tggccctggg 13080
tctgtgggaa ttgtttgcag cctttggcag tgggacacag ccagtgcggc cctggtgctc 13140
ctgtggacgg ccccgcctgg ctaccccagg gggtgtgcag gtaccttatc cagaaagtct 13200
tccaggaggc aggccttcag ggggagctgg gccaccctct tgggccaggt ggcgttcagg 13260
tgcatgagga cggctgggta gtcgtccctg gagatgctct gctctgggaa gtcaaagtcg 13320
ttgatgaggc tgaggatgtt tctgcgggga cagagaggac tgtgggctca tggccctggt 13380
tcccaaacac atccagagaa acccttggtt ggtgctgcga agcacagcat ccctgggagg 13440
gctgggcccc tcctgcccct gttgggatcg tgtgggcccc tcctgcccct gttgggatcg 13500
tgtggacgcc tcctgcccct gttgggatcg tgtggacgcc tccctgcccc tgttgggatc 13560
gtgtggacgc ctacctgccc ctgttgggat cgtgtggacg cctcctgc 13608
<210> 4
<211> 599
<212> PRT
<213> Mus musculus
<400> 4
Met Ala Gln Ala Ser Gly Met Asp Pro Leu Val Asp Ile Glu Asp Glu
1 5 10 15
Arg Pro Lys Trp Asp Asn Lys Leu Gln Tyr Leu Leu Ser Cys Ile Gly
20 25 30
Phe Ala Val Gly Leu Gly Asn Ile Trp Arg Phe Pro Tyr Leu Cys Gln
35 40 45
Thr His Gly Gly Gly Ala Phe Leu Ile Pro Tyr Phe Ile Ala Leu Val
50 55 60
Phe Glu Gly Ile Pro Leu Phe Tyr Ile Glu Leu Ala Ile Gly Gln Arg
65 70 75 80
Leu Arg Arg Gly Ser Ile Gly Val Trp Lys Thr Ile Ser Pro Tyr Leu
85 90 95
Gly Gly Val Gly Leu Gly Cys Phe Ser Val Ser Phe Leu Val Ser Leu
6
CA 02433579 2003-07-02
WO 02/053741 PCT/US02/00111
100 105 110
Tyr Tyr Asn Thr Val Leu Leu Trp Val Leu Trp Phe Phe Leu Asn Ser
115 120 125
Phe Gln His Pro Leu Pro Trp Ser Thr Cys Pro Leu Asp Leu Asn Arg
130 135 140 _
Thr Gly Phe Val Gln Glu Cys Gln Ser Ser Gly Thr Val Ser Tyr Phe
145 150 155 160
Trp Tyr Arg Gln Thr Leu Asn Ile Thr Ser Asp Ile Ser Asn Thr Gly
165 170 175
Thr Ile Gln Trp Lys Leu Phe Leu Cys Leu Val Ala Cys Trp Ser Thr ,.
180 185 190
Val Tyr Leu Cys Val Ile Arg Gly Ile Glu Ser Thr Gly Lys Val Ile
195 200 205
Tyr Phe Thr Ala Leu Phe Pro Tyr Leu Val Leu Thr Ile Phe Leu Ile
210 215 220
Arg Gly Leu Thr Leu Pro Gly Ala Thr Glu Gly Leu Ile Tyr Leu Phe
225 230 235 240
Thr Pro Asn Met Lys Thr Leu Gln Asn Pro Arg Val Trp Leu Asp Ala
245 250 255
Ala Thr Gln Ile Phe Phe Ser Leu Ser Leu Ala Phe Gly Gly His Ile
260 265 270
Ala Phe Ala Ser Tyr Asn Pro Pro Arg Asn Asn Cys Glu Lys Asp Ala
275 280 285
Val Ile Ile Ala Leu Val Asn Ser Met Thr Ser Leu Tyr Ala Ser Ile
290 295 300
Ala Ile Phe Ser Val Met Gly Phe Lys Ala Ser Asn Asp Tyr Gly Arg
305 310 315 320
Cys Leu Asp Arg Asn Ile Leu Ser Leu Ile Asn Glu Phe Asp Leu Pro
325 330 335
Glu Leu Ser Ile Ser Arg Asp Glu Tyr Pro Ser Val Leu Met Tyr Leu
340 345 350
Asn Ala Thr Gln Thr Ala Arg Val Ala Gln Leu Pro Leu Lys Thr Cys
355 360 365
His Leu Glu Asp Phe Leu Asp Lys Ser Ala Ser Gly Pro Gly Leu Ala
370 375 380
Phe Ile Val Phe Thr Glu Ala Val Leu His Met Pro Gly Ala Ser Val
385 390 395 400
Trp Ser Val Leu Phe Phe Gly Met Leu Phe Thr Leu Gly Leu Ser Ser
405 410 415
Met Phe Gly Asn Met Glu Gly Val Ile Thr Pro Leu Leu Asp Met Gly
420 425 430
Ile Leu Pro Lys Gly Ile Pro Lys Glu Val Met Thr Gly Val Ile Cys
435 440 445
Phe Ala Cys Phe Leu Ser Ala Ile Cys Phe Thr Leu Gln Ser Gly Gly
450 455 460
Tyr Trp Leu Glu Ile Phe Asp Ser Phe Ala Ala Ser Leu Asn Leu Ile
465 470 475 480
Ile Phe Ala Phe Met Glu Val Val Gly Val Ile His Ile Tyr Gly Met
485 490 495
Lys Arg Phe Cys Asp Asp Ile Glu Trp Met Thr Gly Arg Arg Pro Gly
500 505 510
Leu Tyr Trp Gln Val Thr Trp Arg Val Val Ser Pro Met Leu Leu Phe
515 520 525
Gly Ile Phe Leu Ser Tyr Ile Val Leu Leu Ile Gln Thr Pro Pro Ser
530 535 540
Tyr Lys Ala Trp Asn Pro Gln Tyr Glu His Phe Pro Ser Arg Glu Glu
595 550 555 560
Lys Phe Tyr Pro Gly Trp Val Gln Val Thr Cys Val Leu Leu Ser Phe
565 570 575
Leu Pro Ser Leu Trp Val Pro Gly Val Ala Leu Ala Gln Leu Leu Ser
580 585 590
Gln Tyr Lys Gln Arg Trp Lys 595
7
